Multi-residue LC-MS analysis

Richard Fussell

CSL York, UK

e-mail: r.fussell@csl.gov.uk

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� Background

� Liquid Chromatography Mass Spectrometry- Options Capabilities and Applications

� Summary

Outline of Presentation

Pesticide Residues :The Analytical Challenge

� Increase the number of pesticides determined in a single multi-residue method

� Lower the detection limits- EU Baby Food Directive 1999/39/EEC –0.01 mg/kg

� Increase the speed of analysis - faster reporting of results to the customer

� Reduce the cost of analysis

But How?8

GC or HPLC approach ?

� What is known about analyte(s)- diverse physico chemical properties- new pesticides tend to be more suitable to LC-MS

� Available methodology- recent developments in acetonitrile extraction more

suited to LC-MS � Available instrumentation

- LC-MS/MS systems becoming more affordable- significant improvement in response during the last decade

GC or HPLC approach ? cont.

Quality aspects- Quantification and confirmation of identity in a single analysis

� Speed of analysis- e.g. recent development of ultra-performance liquid

chromatography (UPLC)

� Costs- expensive, complex and high maintenance

Quattro Ultima Pt with Alliance 2695

Modern LC-MS

� Chromatographic separation� Production of gas-phase ions� Ionisation at atmospheric pressure (API)

� Atmospheric pressure chemical ionisation� Electrospray� Photoionisation

� Transfer (sampling) of ions into MS vacuum� Manipulation of ions, in a vacuum, with electric and

magnetic fieldsto………

� Determine their mass-to-charge ratios (m/z)and…….

� Measure relative abundances of ions of differing m/z

Quattro Ultima SchematicSlide courtesy of Waters

Pesticide Monitoring

� A strategy of Multi-residue GC-MS and LC-MS required for comprehensive screening

� LC-MS becoming more important

� Trend is to transfer pesticides from GC-MS to LC-MS.

But using which LC-MS techniques?

LC-MS Instrumentation

� LC-MS (quadrupole)

� LC-MS/MS (ion trap)

� LC-MS/MS (tandem quadrupole)

� Hybrid instruments

� LC-ToFMS

ES mass spectrum of chlormequat

+Q1: 10 MCA scans from S002.wiff Max. 2.3e7 cps.

50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125m/z, amu

0.0

1.0e6

2.0e6

3.0e6

4.0e6

5.0e6

6.0e6

7.0e6

8.0e6

9.0e6

1.0e7

1.1e7

1.2e7

1.3e7

1.4e7

1.5e7

1.6e7

1.7e7

1.8e7

1.9e7

2.0e7

2.1e7

2.2e72.3e7

Intens

ity, c

ps

122.1

124.0

64.2

123.0104.1 119.155.0 115.083.1 85.062.8 88.2 101.170.9 105.091.074.161.2

Chlorineisotopes

CH2

Cl CH2

N

CH3

CH3

CH3 Cl-

+

Quaternary ammonium m/z 122

HPLC-MS - m/z 122

6 .00 7.00 8 .00 9 .00 10 .00 11.00Tim e13

100

%

lack of selectivityand sensitivity

� monitor pseudomolecular ions using SIM

LC-MS and LC-MS/MS

� But LC-MS has insufficient selectivity for multiresidue analysis of crude extracts – needs MS/MS

� For MS/MS need to induce fragmentation

� Collisionally induced dissociation (CID)- in the collision cell of a tandem type instrument or in

an ion trap device� Product ion spectra (MS/MS spectra)

N

CH3

CH3

CH2

+

NCH3

CH3

CH3

CH2CH2Cl+

m/z 124

m/z 58

35

37

Chlormequat - CID

N

CH3

CH3

CH2

+

NCH3

CH3

CH3

CH2CH2Cl+ 35

m/z 58

m/z 122

LC-MS/MS chromatograms showing detection of chlormequat

6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0

Time, min

0

5

10

15

20

25

30

35

40

45

50

55

60

ity, cps

6.05

8.31

8.166.04 8.44 10.40

11.496.98 9.39 10.797.997.63 10.31

6.57 11.319.48 10.226.95 10.966.26 8.517.41 9.36

7.326.85 8.82 10.039.728.91 11.90

6.896.44

6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0

Time, min

0

100

200

300

400

500

600

700

800

900

1000

1100

1200

1300

1400

1500

1600

1700

1800

1900

2000

ity, cps

8.00

8.07

7.92

8.23

7.72

8.48

8.56

8.82

9.38

Blank

0.005 mg/kgAPI 2000

m/z 122>58

m/z 124>58

Use ratio of abundancesto confirm identity

LC-MS/MS (tandem quadrupole)

� Improved selectivity & sensitivity in MRM mode

� Official acceptance as confirmation technique

� More robust quantification compared to Ion traps

But

� Limited to target compounds

� Not truly multi-residue (peak capacity/ionisation)

� Can still be subject to isobaric interferences matrix effects (e.g. suppression), adduct formation

� Chromatography still important

HPLC-MS : Multi-Residue Methods 1996

5.00 10.00 15.00 20.00Time0

100

%

� ES+� Ethyl acetate with HPGPC or SPE clean-up & concn

� 30 carbamates, their metabolites and benzimidazoles � LOQ = 0.05 – 0.1mg/kg

HPLC-MS/MS : Multi-Residue Methods 2006

5 10 15 20 25 30 35 40T ime, m in

0.0

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

1.2e5

1.4e5

1.6e5

1.8e5

2.0e5

2.2e5

2.4e5

2.6e5

2.8e5

3.0e5

3.2e5

3.4e5

3.5e5

17.14

21.9818.02

� ES+ � Reversed Phase HPLC� QuEChERS acetonitrile extraction – no clean-up� >100 pesticides � LOQ = 0.01 mg/kg

QuEChERS HPLC-MS/MS – Increased Scope

Typically include pesticides with diverse characteristics

� Basic and acidic (chromatography?)

� Polar pesticides (column selection)

� Pesticides previously analysed by single residue methods

� Pesticides that may degrade using GC-MS

� Pesticides that require derivatisation or chemical conversion to single moiety for GC analysis

But some pesticides still require SRM or other technique e.g. GC-MS

� Klein J & Alder L. J. AOAC Int., 2003; 86, 1015-1037- methanol-water extraction, approx 100 pesticides

� Jansson C, et al, J. Chromatogr. A, 2003; 1023, 93-104- ethyl acetate, 57 pesticide compounds

� Hetherton et al , J. Rapid Communications Mass Spectrometry, 2004; 18, 2443-2450- acetonitrile, 73 pesticide compounds

� Lehotay et al, J. AOAC Int., 2005; 886, 595 - 614- acetonitrile, 144 pesticide compounds

Multi-residue LC-MS/MS methods

LC-MS/MS considerations/limitations

Multi-residue methods are always a compromise

� Electrospray or APCI?

� Positive or negative mode – polarity switching?

� Availability of analogue standards

� Matrix-matched calibration to minimise suppression and enhancement by matrix components

In practice need more than one analysis but ES+ provides best response for more compounds

UPLC-MS/MS

� Waters Premier XE� Acquity UPLC BEH C18 Column, 2.1 x 100mm,

1.7µm at 40°C� >50 pesticides < 5 mins

UPLC – MS/MS with Polarity switching

Cyprodinil, +ve

Fenoxycarb, +ve

Diflubenzuron, -ve

� + ve and –ve mode pesticides in a single chromatographic run.�Narrow peaks >s/n�Resolution of geometric isomers�Allows higher throughput (samples per day/per instrument)�Instead of switching perform short runs in either + ve or –ve mode

�Chromatography compromised for late eluting pesticides

Capillary

HPLC inlet Nebulizer

Drying gas

Corona needle

ESI Zone

APCI Zone

Thermalcontainer

IR emitters

Chargingelectrode

Reversing electrode

Sensor for vapor temperature

APCI counterelectrode

Overview of the Multimode SourceSlide courtesy of Agilent

�Simultaneous ES and APCI

�Compatible with +ve/-ve polarity switching

�Applied to pesticides

LC-MS/MS weaknesses

� Not all pesticides will be detected

� High risk that non compliances will be missed

� Strategic data on the legal and illegal use of pesticides will not be captured

Accurate mass TOFMS – potential advantages

� Increase in the number of pesticides screened in a single analysis (at least not so limited by the acquisition programme)- some limitations –ionisation etc.- number also limited by solubility during extraction- clean-up?- chromatography, pKa etc.- LOQ ? - 0.01 mg/kg may not be possible

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LC-TOF Schematic

Two Stage IonMirror

5 Stage Vacuum System

Agilent Orthogonal Spray Source(s)

Optically CoupledIon Detector

Beam cooling and guidance

Effective Flight PathLength of 2.0m Low-expansion

Flight Tube

Slide courtesy of Agilent

HPLC- TOFMS

� Compatible with fast chromatography

� High sensitivity over wide mass range

� Detection of ‘unknowns’?

� Retrospective searching

but

� Lower sensitivity than high spec triple quads

� Require new approaches to calibration

� Linearity

� Time consuming data processing

Hybrid Instruments

� QTOF - Hybrid MS/MS with quadrupoleprecursor selection and time-of-flight product ion “scanning”

� Structural Information

� QTRAP – Simultaneous Quantification and Qualitative Confirmation of 300 pesticides

� More expensive than triple quad MS/MS� Not yet used routinely

LC - MS Summary

� Conventional HPLC now of limited use

� LCMS especially LC-MS/MS now affordable - easier to use- less clean-up- quantification & confirmation in single analysis - up to 200 pesticides per analysis

� TOF MS Increasing acceptance of screening approach

Thank you for your attention