Orbitrap Technology: New Frontiers in Pesticide Residue Analysis

Michal Godula

Special Solutions Center Europe

Thermo Scientific

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Global Scale

We Are The World Leader in Serving Science

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• No compromise MS, MS/MS and MSn

• Speed, sensitivity, ultrahigh resolution, precise mass accuracy, and dynamic range - at the same time

Orbitrap: The Performance Leader since 2006

What Orbitrap provides

Unmatched ultrahigh resolution, accurate mass performance

Which applications are optimized• Virtually all molecular analyses:

accurate identification, structural analysis, and quantification of organic molecules, lipids, carbohydrates, peptides & proteins in complex mixtures

• Proteomics, drug discovery, toxicology, forensics, environmental, food safety and many more

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Orbitrap FTMS

Makarov A. Anal. Chem. 2000, 72, 1156-1162.

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Orbitrap Mass Analyzer Features

• Fundamental difference to other HRAM instruments• Parameter measured is frequency, not time/voltage/current• Resolution allows more accurate m/z determination• Less prone to ambient conditions changes• Usually stable within <2 ppm during several days• No need for lock mass in “routine work”• Small footprint• Easy to setup

How accurate is your mass?

Mass accuracy

610/

true

truemeas

m

mmzm

• Quadrupole MS

• Orbitrap MSTOF MS

ppmzm 20010500

0.5001.500/ 6

ppmzm 21010314.500

10214.50010314.500/ 6

Selectivity increases with higher mass accuracy

7.2 7.4 7.6 7.8 8.0 8.2 8.4 8.6 8.8 9.0 9.2 9.4 9.6 9.8 10.0 10.2 10.4 10.6Time (min)

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RT: 8.45

9.298.728.678.99

8.789.118.217.85 8.838.137.627.59 7.81 8.10 8.357.38

7.337.27 9.46 10.6110.509.50 10.3410.169.64 10.09

100 ppm

20 ppm

2 ppm

7.2 7.4 7.6 7.8 8.0 8.2 8.4 8.6 8.8 9.0 9.2 9.4 9.6 9.8 10.0 10.2 10.4 10.6Time (min)

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RT: 8.45

8.217.62 7.857.598.257.407.37 7.81 7.92 7.99

8.617.30 10.617.26 10.508.88 10.318.67 9.11 10.1610.099.46

7.2 7.4 7.6 7.8 8.0 8.2 8.4 8.6 8.8 9.0 9.2 9.4 9.6 9.8 10.0 10.2 10.4 10.6Time (min)

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RT: 8.45

8.557.997.49 7.917.76 10.617.417.26 10.5010.3110.167.67 9.46

NL: 1.20E5m/z= 484.25313-484.25507 F: FTMS {1,0} + p APCI Full ms [300.00-850.00] MS pivo_5_UH

*Zachariasova M et al, Anal. Chim. Acta

Resolution (resolving power)

• Resolution

m

mR

m (FWHM)

m 10%

8336.0

500

m

mR• Quadrupole MS

• Orbitrap MS 100000005.0

500

m

mR

Does resolution matter?

RAFA 2013: Łukasz Rajski, María del Mar Gómez Ramos, Amadeo R. Fernández-Alba; EURL for Pesticide Residues in Fruits and Vegetables. Pesticide Residue Research Group. University of Almeria, Spain. e-mail: amadeo@ual.es

Q Exactive MS - a 3D view

Hyperbolic Quadrupole Mass filter

HCD Cell

Orbitrap Mass Analyzer

C-Trap

RF-Lens

Q Exactive FS-ddMS2 screening process

Inclusion List

Ion Accumulation

Full Scan Orbitrap Analysis

Precursor Selection

Precursor accumulation

Fragmentation in HCD

MS2 Orbitrap Analysis

Full Scan MS Data: Screening/Quantitation

Full Scan MS/MS Data:Confirmation

Multi-residue screening by Q Exactive LC-MS

• Complete solution for quantitiative analysis of LC amenable compounds• Implementation method for most relevant pesticides/mycotoxins and

pyrrolizidine alkaloids covering different chemistries• In 3 relevant matrices• Critical evaluation of established method performance criteria vs. current

regulation

strawberry leek wheat flour

Quechers extraction

10 g of homogenized sample is weight into Quechers extraction tube

+ 20 mL of water

+ 10 mL of ACN

Centrifugation 5 min @ 5000 rpm

Analysis

shaking 10 min

Ext

ract

ion

UHPLC instrumental method

Thermo ScientificTM UltiMate® 3000 RSLC : • column: Accucore aQ 100 mm x 2.1 mm x 2.6 µm • Column oven T: 25 °C • Injection Volume: 1 µL• Mobile phase A: Water/MeOH (98:2) 5mM Ammonium

formate, 0.1% FA• Mobile phase B: Water/MeOH (2:98) 5mM Ammonium

formate, 0.1% FA• Flow rate: 300 µL/min

Q Exactive Conditions – ddMS2 experiment

• Q-ExactiveTM MS conditions: Spray voltage 3800V, Capillary temperature 295°C, sheath gas 32 au, Aux gas 7 au, S-lens RF level 55

• Full MS/data dependent MS2

• full MS:resolution 70,000 FWHM

scan range 120-1200 m/z

• ddMS2: resolution 35,000 FWHM

Inclusion list on

isolation 1 m/z

exclusion 5 s

List of 556 target analytes

1234567891011121314151617181920212223242526 … … …556

Isotopes

Fragments

LibrarySpectrum

Chromatogram

Target analytes

TraceFinderTM Screening Data Review

Thermo EFS Screening Database: 1729 substances

• Targeted screening in “known RT” compounds• Untargeted in the rest of database (RT not known)

Screening Example – Acetamiprid in honey sample

Acetamiprid confirmation

Isotopic Pattern

+ Main fragment

Acetamiprid details

Standard 50 ug/kg

Sample – determined concentration 5.3 ug/kg

SampleMS2 spectra

Standard MS2 spectra

Isotopic pattern fitting is influenced by resolution

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Q Exactive Focus: Introducing vDIA

• Variable Data independent acquisition (vDIA) acquires MS2 spectra systematically and independent of previously acquired scans

• You obtain:• complete record of data

• very good reproducibility

• high throughput comprehensive quantification acquisition methods with quantitative information

• no requirement for detailed sample knowledge prior to data acquisition

• Your routinely acquired data is ready to go for any non-target screening approach

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Variable DIA – alternative to All Ions Fragmentation

Presented by P Zomer, RIKILT at EPRW 2014:

Traditional AIF

Variable Data Independent Analysis

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AIF vs. vDIA

AIF

vDIA

Oxacillin in Pig Kidney @ 2 ppb background

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Running 380 Compounds with the Q Exactive Focus

Data Dependent-MS2 vDIA

Quan/Qual Quantitation Targeted Screening

# of targeted compounds

Total: 380345 Pesticides, 35 Mycotoxins and Alkaloids

Selectivity Retention time, fragment ions, ion ratios, isotope ratio, library match

Linearity – working range [ppb]

1-500 (6 levels and 2 replicates)

Matrix Black Tea/Honey

Sample Prep Buffered QuEChERS method clean-up made by MgSO4, PSA and C18

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vDIA sensitivity in honey

80%

20%

Number of compounds with <1ppb sensitivity

Peaks in honey sample spiked to 1ppb

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345 target pesticides in tea matrix

Data still being processed

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A New Chapter in GC-MS

Thermo Scientific Q Exactive GC Hybrid Quadrupole-Orbitrap GC-MS/MS System

Offering Unprecedented Depth in GC-MS Analysis

• Hugely powerful HR/AM performance for GC-MS

• Uncompromised quantitative and qualitative capability

• Reliable, robust and easy to operate

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Bringing GC and Orbitrap Technology Together

C-TRAP

HCD Cell

AQT Quadrupole

Orbitrap Mass Analyzer

BentFlatapole

ExtractaBrite™ Ion Source

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Breakthrough in GC-MS Performance

Highest selectivity and confidence with high resolving power

Unrivalled Resolving Power

• Up to 120k @ m/z 200

• Allows excellent selectivity in complex matrix, maintaining confidence in detection and identification

• Drives highly resolved data sets and high capacity peak detection

• Fast scanning Orbitrap for well defined, reproducible GC peaks

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Breakthrough in GC-MS Performance

Highest selectivity and confidence with high resolving power

Chlorprophram

• 10 ng/g QuEChERS extract of leek

• Full scan with resolving power of >30k (FWHM @ m/z 200) provides interference free detection

• Excellent mass accuracy for confident identification

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Pesticides screening

High resolving power crucial to avoid false negatives

False negatives at 15,000 RP

Secure screening

• 5 ppm identification threshold

• Higher resolving power allows confident detection and identification

• Mid- resolution of 15k causes false negatives

• Q Exactive GC routinely operates at 60k (FWHM @ m/z 200)

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Breakthrough in GC-MS Performance

Highest selectivity and confidence with high resolving power

XIC of dieldrin in baby food (m/z 262.85642) 100ppb

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3

4

5

6

7

89

10

11

12

13

14

1516 17

60,000 RP (m/z 200) 2.4 s peak

Fast acquisitions

• Important for accurate profiling of narrow GC peaks

• Full scan with resolving power of 60 (FWHM @ m/z 200) generates 17 scans

• Fast enough for GC!

17 scans

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Scan to-scan

XIC of dieldrin in baby food (m/z 262.85642) 100ppb

60,000 RP (m/z 200) 2.4 s peak

Breakthrough in GC-MS Performance

Consistently excellent mass accuracy

< 1ppm (internal)

< 3ppm (external)

• Scan-to-scan

• Low level in matrix

• Over full mass range

• Over full concentration range

• No need for continuously calibrating in sequence

0.70.7

0.6

0.5

0.3

0.6

0.5

0.3

0.3

0.3

0.5

0.6

0.6

0.70.5

0.5 ppm RMS

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Breakthrough in GC-MS Performance

Consistently excellent mass accuracy

Mass accuracy over >6 orders

• Hexachlorobenzene

• 0.2 – 250,000 ng/mL

• Mass accuracy within 1 ppm

• Orbitrap spectral fidelity independent of concentration

Over full concentration range

Data courtesy of Hans Mol and Marc Tienstra, RIKILT

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Is it sensitive enough? Q Exactive GC vs. Triple Q

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Breakthrough in GC-MS Performance

Unprecedented quantitation power for HR/AM GC-MS

SRMFull Scan

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Compound Discovery and Identification

Tracefinder: automated compound detection and identification

1. Peak detection

2. Spectral deconvolution

3. Library searching (e.g. NIST, Wiley, custom)

4. % TIC explained using elemental composition and accurate mass filtering

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Automated Workflow for Accurate Mass Confirmation

Unknown Identification Workflow

Spectral Deconvolution on EI HRAM

Data

Spectral Library SearchScoring

AdditionalHigh

Resolution Scoring

m/z = 275.11298

C11H23O4Si2m/z = 275.11294

m/z = 275.11298

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Pesticides screening

Horse feed detection limit

100 % detected at MRL levelMajority at ½ MRL

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Pesticides screening

Quantitation

Fenpropimorph calibration curve in leek matrix.

• For all pesticides, the coefficient of determination (R2) was >0.99 with an average value of R2 = 0.997

Curve from 0.5 to 50 ng/g (triplicate at each level)

R2=0.9999

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Complete solution for total pesticide analysis

Q Exactive GC Hybrid Quadrupole-Orbitrap GC-MS/MS System

Q Exactive FocusHybrid Quadrupole-Orbitrap LC-MS/MS System