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Transcript of Ion-mobility Tof Mass Spectrometry - Waters · ©2012 Waters Corporation 1 Ion-mobility Tof Mass...

2012 Waters Corporation 1

Ion-mobility Tof Mass Spectrometry

A new dimension for analyte identification in crude extracts

2012 Waters Corporation 2

Overview Introduction and background Residue analysis current challenges? Conventional HR-MS screening A new dimension for identification- QToF with ion mobility

separation (HDMS) Ion-mobility principle

A tool for analyte identification- examples Identification of veterinary drug residues as environmental

pollutants in waste water samples Observation of multiple sites of intra-molecular protonation Reduction of spectral complexity

Summary and conclusions Future prospects?

2012 Waters Corporation 3

Residue Analysis current challenges... Occurrence of new residues

Metabolites, biotransformation and degradation products... Foodstuffs are sourced from throughout the world using ingredients

from multiple countries

More stringent Regulatory requirements Screen for larger numbers of compounds at lower concentrations in

complex matrices Additional sensitivity and performance requirements?

Increasing consumer awareness Melamine in milk products, synthetic hormones in meat, illegal dyes in

fish, antibiotics in honey

2012 Waters Corporation 4

Conventional HR-MS screening?

High resolution mass spectrometry (HR-MS) has gained in popularity as a screening tool

Ability to perform non-targeted analysis

Freedom to measure compounds without prior compound specific tuning

Ability to perform historical (retrospective) data review Capable of performing structural elucidations of unknowns or suspected

compounds

Ability to perform full scan spectral analysis Providing greater insight into the composition of a complex sample

Increased specificity in complex matrices Accurate mass, diagnostic fragment ions

2012 Waters Corporation 5

Ion Mobility - A New Dimension for HR-MS

SYNAPT G2 HDMS (QTof plus ion mobility)

A new dimension for method development and extract characterisation

Ion mobility allows compounds not previously separated and recorded with other LC-MS approaches to be distinguished

Isobaric compounds, isomers, conformers separated by ion mobility

Cleaner MS spectra - data interpretation easier

Observations about the molecular properties

G2 Qtof platform Ionisation flexibility (universal source) ESI, APCI, APPI, APGC, MALDI, DESI, DART

Detect ionised compounds at high and trace levels in complex matrices Compatible with high resolution UPLC In spectrum dynamic range Comprehensive data acquisition with accurate mass data for precursor

and product ions) MSE Confidence in mass accuracy and stability

Analyse and interpret data Variety of application managers to support a variety of workflows

Obtain repeatable results IntelliStart, QuanTof technology

2012 Waters Corporation 6

Data acquisition using MSE

UPLC-MSE is a data independent parallel process that occurs in the collision cell

The instrument is operated in an alternative scanning mode providing two MS scan functions for data acquisition in one analytical run Function 1 = low collision energy (precursor ions) Function 2 =

high energy (fragment ions)

DATA ACQUISITION Acquire data-independent MSE Data

MS/MS MRM Experiment?

2012 Waters Corporation 7

Schematic of the instrument

SYNAPT G2-S High Definition MS (HDMS)

Size

Shape

Charge

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Ion-Mobility Principle

Small and compact rapid acceleration

Large, extended

molecule = longer drift

time

80 year old concept (C.F. Powell, 1932) Ions race the most mobile reach the detector first Separation is driven by electric fields not under vacuum

2012 Waters Corporation 9

Ion mobility - a new dimension for analyte identification?

Identification of fluoroquinolone residues in waste water samples

Collaboration with Institut Catal de Recerca de l'Aigua (ICRA)

2012 Waters Corporation 10

Database search = ciprofloxacin

identified in effluent sample

Total ion current view

Analysis of veterinary drug residues in spiked waste water conventional targeted QTof MS

Conventional extracted mass chromatogram m/z 332 in MassLynx shows one peak at 1.92 min

2012 Waters Corporation 11

Using Ion Mobility - Drift time vs retention time

Retention time (mins)

Dri

ft t

ime

(ms)

TOF MS BPI spectrum

Ion mobility spectrum

2012 Waters Corporation 12

View of orthogonal mobility separation using Driftscope

4947_214.raw:1

4947_214.raw : 1Extracted mass chromatogram M/Z 332 one peak at 1.92 mins

Mobility Data Two components peak detected for M/Z 332 at 1.92 mins

M/Z 332 Two components two Drift times

Two components same exact mass

2012 Waters Corporation 13

3D view of mobility separated co-eluting isobaric masses at 1.92 mins

Ciprofloxacin (M+H=m/z 332.1410)

F

O

OH

O

N N

N

H

H+

2012 Waters Corporation 14

MSe Data viewer application manager Isobaric masses at retention time 1.92 minutes, with different Drift Times filtered from 1456 components

BPI

MS

MSE

2012 Waters Corporation 15

Exact mass and elemental composition for mobility separated isobaric masses at 1.92 mins - 1

Peak at drift time 4.9 ms

C17H19N3O3F Error= 1.8 ppm

F

O

OH

O

N N

N

H

H+

Confidence in elemental composition assignment

2012 Waters Corporation 16

Exact mass and elemental composition for mobility separated isobaric masses at 1.92 mins - 2

Peak at drift time 4.3 ms

C17H19N3O3F Error= 3.0 ppm

F

O

OH

O

N N

N

H

H+

Confidence in elemental composition assignment

2012 Waters Corporation 17

Ion mobility - a new dimension for analyte identification?

Discovery of multiple sites of intra-molecular protonation and different fragmentation

patterns Collaboration with RnAssays B.V.

2012 Waters Corporation 18

UPLC IMS MSe BPI for a mixed solvent standard

Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50

%

0

100SynaptG2_20122901_039 Sm (SG, 1x1) 1: TOF MS ES+

BPI5.32e4

2.29

2.02

0.33

1.99

2.38

2.59

3.79

3.04

2.84

9.25

9.13

3.946.69

4.50 4.72

4.896.74

8.587.94

9.37

Ciprofloxacin Generic gradient conditions - mixed

solvent standard containing 25 antibiotic compounds

2012 Waters Corporation 19

Conventional accurate mass spectrum for ciprofloxacin at Rt 2.2 mins

m/z140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340

%

0

100SynaptG2_20122901_039 468 (2.194) AM2 (Ar,18000.0,556.28,0.00,LS 5); ABS; Cm (464:473) 1: TOF MS ES+

2.32e5332.1410

157.5692

144.9824

158.0701166.5742 274.0992

167.0751215.1261186.9564 203.0422

219.8978245.1370241.8860

251.9137 262.8581 279.0943 288.1510 320.1388314.1290

333.1437

334.1464

336.1539

Ciprofloxacin (M+H=m/z 332.1410)

F

O

OH

O

N N

N

H

H+0 ppm

Characteristic spectrum doubly charged species at m/z 166.5

2012 Waters Corporation 20

Expanded IMS MSe BPI for a mixed solvent standard

Time1.80 1.90 2.00 2.10 2.20 2.30 2.40 2.50 2.60 2.70 2.80 2.90 3.00 3.10 3.20 3.30 3.40 3.50 3.60 3.70 3.80 3.90 4.00 4.10

%

0

100SynaptG2_20122901_039 Sm (SG, 1x1) 1: TOF MS ES+

BPI5.32e4

2.29

2.02

1.99

2.06 2.19

2.13

2.26

2.38

2.32

2.59

2.56

3.79

3.04

2.84

2.71

3.94

SynaptG2_20120102_437.raw : 1

Orthogonal Ion Mobility Separation

Dri

ft T

ime

(ms)

Retention Time (mins)

Norfloxacin, sarafloxacin, difloxacin, enrofloxacin , enoxacin, perfloxacin, ofloxacin, ciprofloxacin, marbofloxacin

Ciprofloxacin

2012 Waters Corporation 21

Ion mobility separation of ciprofloxacin - observation of two protonated species

Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50

%

0

100SynaptG2_20120102_233 2: TOF MS ES+

332.047_334.391 0.0500Da4.32e5

5.48

4.34

F

O

OH

O

N N

N+

H

H

F

O

O+

O

N N

N

H

HH

Dt = 4.34 ms Dt = 5.48 ms

Drift time (ms)

2012 Waters Corporation 22

MSe IMS ciprofloxacin and Conventional Spectrum

m/z100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500

%

0

100

m/z100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500

%

0

100

m/z100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500

%

0

100SynaptG2_20120102_290 238 (2.227) AM2 (Ar,18000.0,556.28,0.00,LS 5); ABS; Cm (237:240) 2: TOF MS ES+

2.32e5314.1323

231.0580

203.0624136.0564116.0512 163.0672

175.0677217.0789

288.1519245.1097

274.0994246.1127 289.1544

332.1421

333.1444

354.1225

SynaptG2_20120102_290_dt_02 76 (4.050) AM2 (Ar,18000.0,556.28,0.00,LS 5); ABS 1: TOF MS ES+ 6.50e4314.1331

231.0592

232.0596288.1513274.0989

315.1338332.1425

333.1440

SynaptG2_20120102_290_dt_02 97 (5.184) AM2 (Ar,18000.0,556.28,0.00,LS 5); ABS 1: TOF MS ES+ 2.98e4288.1526

245.1099

204.0704189.0469175.0712

231.0940217.0787

246.1131 268.1457

332.1420

289.1537

314.1308

333.1425

334.1440

Mobility separated Dt 4.34 ms

Mobility separated Dt 5.48 ms

Conventional spectrum

Observation of different fragmentation pathways

m/z 231

2012 Waters Corporation 23

Ion mobility - a new dimension for analyte identification?

Reducing spectral complexity Multi-residue analysis of crude porcine tissue

extracts

2012 Waters Corporation 24

Conventional UPLC MS data 25 compound spiked porcine muscle extract -1

N

OH

O

O

CH3

O

O

EMC Oxolinic acid m/z 262 @ Rt 3.03 mins

Total ion current

2012 Waters Corporation 25

Using ion mobility - matrix removal

Co-eluting components

Mobility separation matrix removed N

OH

O

O

CH3

O

O

Oxolinic acid background [email protected] Rt 3.03 mins

2012 Waters Corporation 26

Conventional UPLC MS data 25 compound spiked porcine muscle extract -2

EMC Oxytetracycline m/z 461 @ Rt 2.17 mins

OH O OHOH

O

CH3OHOH

NCH3CH3

NH2

O

OH

Total ion current

2012 Waters Corporation 27

Using ion mobility - matrix removal

Co-eluting components

Mobility separated oxytetracycline matrix removed

OH O OHOH

O

CH3OHOH

NCH3CH3

NH2

O

OH

Oxytetracycine background [email protected] Rt 2.17 mins

2012 Waters Corporation 28

S/Ns obtained at MRL concentrations in spiked porcine tissue extract

Flumequine

Ciprofloxacin

Oxytetracycline

OH O OHOH

O

CH3OHOH

NCH3CH3

NH2

O

OH

NNH

N

O

OH

OF

N

CH3

O

OH

OF

2012 Waters Corporation 29

Flumequine UPLC IMS MSe Porcine tissue spiked at 0.5x MRL

E:\2012_RNASSAYS G2S_MMcC.PRO\Data\SynaptG2S_ RNASSAYS_21MARCH2012_012.raw:1

SynaptG2S_ RNASSAYS_21MARCH2012_012.raw : 1

2012 Waters Corporation 30

Flumequine UPLC IMS MSe Porcine tissue spiked at 0.5x MRL

SynaptG2S_ RNASSAYS_21MARCH2012_012.raw:1

SynaptG2S_ RNASSAYS_21MARCH2012_012.raw : 1

2012 Waters Corporation 31

Summary and Conclusions Using IMS MSe it has been possible to observe; Separation of different intra-molecular protonated species (IMS) Multiple sites of protonation (IMS) Different fragmentation pathways (MSe )

IMS can effectively resolve analyte peaks from matrix interferences

and remove the need for complex sample clean-up & chromatographic separations Generate single component MS and MSe fragmentation spectra Crude extracts from high-throughput screening assays are

compatible with quantitative / confirmatory LC-MS/MS

Initial results have shown UPLC IMS MSe to be applicable to veterinary drug residue analysis at 0.5x MRL

2012 Waters Corporation 32

Future Prospects?

IMS drift time can be used to calculate collision cross section (CCS) areas () for compounds

CCS values are an inherent property of that

compound

In addition to retention time, precursor ion accurate mass, accurate mass fragmentation spectra, CCS values can be used as an identification point

A new identification point revision to 2002/657/EC?

2012 Waters Corporation 33

Acknowledgements Mike McCullagh Waters, Manchester, UK Antonietta Gledhill - Waters, Manchester, UK Joanne Williams - Waters, Manchester, UK Jon Williams - Waters, Manchester, UK Dominic Roberts Waters, Manchester, UK Aldert Bergwerff RnAssays, Utrecht, Netherlands Wouter de Keizer - RnAssays, Utrecht, Netherlands Institut Catal de Recerca de l'Aigua (ICRA)

Thank you for your attention!

Ion-mobility Tof Mass Spectrometry A new dimension for analyte identification in crude extractsOverviewResidue Analysis current challenges...Conventional HR-MS screening? Ion Mobility - A New Dimension for HR-MSData acquisition using MSESYNAPT G2-S High Definition MS (HDMS)Ion-Mobility PrincipleIon mobility - a new dimension for analyte identification?Using Ion Mobility - Drift time vs retention timeView of orthogonal mobility separation using Driftscope3D view of mobility separated co-eluting isobaric masses at 1.92 minsMSe Data viewer application managerExact mass and elemental composition for mobility separated isobaric masses at 1.92 mins - 1Exact mass and elemental composition for mobility separated isobaric masses at 1.92 mins - 2Ion mobility - a new dimension for analyte identification?UPLC IMS MSe BPI for a mixed solvent standardConventional accurate mass spectrum for ciprofloxacin at Rt 2.2 minsExpanded IMS MSe BPI for a mixed solvent standardIon mobility separation of ciprofloxacin - observation of two protonated speciesMSe IMS ciprofloxacin and Conventional SpectrumIon mobility - a new dimension for analyte identification?Conventional UPLC MS data 25 compound spiked porcine muscle extract -1Using ion mobility - matrix removalConventional UPLC MS data 25 compound spiked porcine muscle extract -2Using ion mobility - matrix removalS/Ns obtained at MRL concentrations in spiked porcine tissue extractFlumequine UPLC IMS MSe Porcine tissue spiked at 0.5x MRLFlumequine UPLC IMS MSe Porcine tissue spiked at 0.5x MRLSummary and ConclusionsFuture Prospects?Acknowledgements