high-resolution enantiomeric separations on a slim im-ms … · 2018. 7. 16. · traveling wave ion...

High-Resolution Enantiomeric Separations on a SLIM IM-MS Platform

Gabe NagyPacific Northwest National Laboratory

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Goal: development of a fast, sensitive, and high-resolution technique for enantiomeric separations

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Current Toolbox for Enantiomeric Separations

What are enantiomers?

Capillary electrophoresis or liquid chromatography-based methods most commonly used

Chiral derivatization: turns enantiomers into potentially resolvable diastereomers

Chiral stationary phases: rely on chiral interactions between enantiomeric analyte and stationary phase

3Ward, T. J.; Ward, K. D. Anal. Chem. 2012, 84, 626−635.

D-aspartic acid L-aspartic acid L-threonine L-serineD-threonine D-serine

Ion Mobility-Mass Spectrometry (IM-MS)-Based Approaches

Attractive alternative: faster than chromatographic methods

No chiral stationary phase supports in IM separations and enantiomers have identical collision cross sections, so need other chiral interactions

Chiral drift gas of S-(+)-2-butanol at atmospheric pressure (limited follow-up work)

Formation of chiral non-covalent gas-phase complexes: poor resolution and resolving power (limited by path length) and sensitivity (limited charge capacity for ion introduction in existing platforms)

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Dwivedi, P.; Wu, C.; Matz, L. M.; Clowers, B. H.; Siems, W. F.; Hill Jr., H. H. Anal. Chem. 2006, 78, 8200−8206.Dodds J. N.; May, J. C.; McLean, J. A. Anal. Chem. 2017, 89, 952−959.Gaye, M. M.; Nagy, G.; Clemmer, D. E.; Pohl, N. L. B. Anal. Chem. 2016, 88, 2335−2344.

Could Structures for Lossless Ion Manipulations (SLIM) help overcome these limitations of existing

IM-MS-based enantioseparations?

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SLIM IM-MS Overview

SLIM enables traveling wave ion mobility (TW IM) separations through the use of electric fields generated by arrays of electrodes (RF and DC potentials) on evenly spaced planar surfaces fabricated with photolithography

6Deng, L.; Ibrahim, Y. M.; Hamid, A. M.; Garimella, S. V. B.; Webb, I. K.; Zheng, X.; Prost, S. A.; Sandoval, J. A.; Norheim, R. V.; Anderson, G. A.; Tolmachev, A. V.; Baker, E. S.; Smith, R. D. Anal. Chem. 2016, 88, 8957−8964.

Ultralong Path Length Separations in SLIM Traveling wave ion mobility (TW IM) separations permit much longer path lengths than drift

tube (DT) IM separations due to their higher voltage constraints

Switch region routes ions either to time-of-flight (TOF) detector or to do another “pass” permitting much longer path length separations than with existing IM platforms (>500 m)

7Deng, L.; Webb, I. K.; Garimella, S. V. B.; Hamid, A. M.; Zheng, X.; Norheim, R. V.; Prost, S. A.; Anderson, G. A.; Sandoval, J. A.; Baker, E. S.; Ibrahim, Y. M.; Smith, R. D. Anal. Chem. 2017, 89, 4628−4634.

Compression Ratio Ion Mobility Programming (CRIMP)

Stuttering or intermittently applied traveling waves in stuttering trap (ST) section allows for ions from traveling trap (TT) bins to be redistributed into smaller number of bins in ST section and thereby compressed

8Deng, L.; Garimella, S. V. B.; Hamid, A. M.; Webb, I. K.; Attah, I. K.; Norheim, R. V.; Prost, S. A.; Zheng, X.; Sandoval, J. A.; Baker, E. S.; Ibrahim, Y. M.; Smith, R. D. Anal. Chem. 2017, 89, 6432−6439.

Peak shape and peak height as a function of compression ratio

CRIMP can successfully “compress” broad initial ion packets

Compression Ratio Ion Mobility Programming (CRIMP)


Liulin Deng

Ion Accumulation in SLIM Module Ion accumulation can be achieved by blocking ions at the interface between the two

TW regions to permit accumulation of >109 ions


What is a potentially suitable chiral modifier to form such non-covalent gas-phase complexes with D/L enantiomer pairs

to permit chiral separation on a SLIM IM-MS platform?

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Cyclodextrins? Previous work has shown their potential as both mobile phase modifiers and LC stationary

phase supports for isomeric and enantiomeric separations

Ability to form host-guest inclusion complexes based on their hydrophobic cavity and hydrophilic exterior

Choice of amino-substituted CDs to have preferred protonation site for positive mode MS and potentially limit number of ion conformations in IM separations that might convolute analyses

12Zhou, J.; Tang, J.; Tang, W. Trends Anal. Chem. 2015, 66, 22−299.

3-amino 3-deoxy α-cyclodextrin (α) 3-amino 3-deoxy β-cyclodextrin (β)

Potential Challenges/Questions for SLIM IM-MS Approach?

Will these complexes form in solution and survive ESI?

Will our traveling wave conditions be gentle enough not to fragment these non-covalent complexes?

Will these complexes survive multiple SLIM passes?

How can a large accumulation region and subsequent CRIMP step be combined to maximize both resolution and sensitivity?

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Complex Formation? Analytically relevant formation efficiency of desired chiral non-covalent complex

Only 2:1 CD:AA host-guest inclusion complexes observed (no 1:1)

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1100 1200 13000

1

m/z

Rel

ativ

e in

tens

ity

1134.3 m/z[β + H]+ and [2β + 2H]2+

1205.8 m/z[2β + Thr + Li + H + H2O]2+

Other peaks:1146.3 m/z: [2β + Li + H + H2O]2+ 1153.3 m/z: [2β + K + H]2+1156.3 m/z: [β + Na]+

Can IM-MS provide any insight into complex formation (i.e. the host-guest inclusion complex)?

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Host-Guest Inclusion Complex Shape? Cyclodextrin dimer more rigid in structure than monomer

Potential insight as to why observed host-guest inclusion complexes exist as 2:1 CD:AA

[2β + 2H]2+ [β + H]+

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Host-Guest Inclusion Complex Shape? Previous literature has shown ‘head-head’ as most stable dimer arrangement due to

intermolecular hydrogen bonding network

Working hypothesis: cyclodextrin dimer encapsulates the amino acid analyte?

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ββ

ββ

β

β

Bonnet, P.; Jaime, C.; Morin-Allory, L. J. Org. Chem. 2001, 66, 689−692.

Tail-Tail Head-Tail Head-Head

Effect of CRIMP on Complexation and Eventual Resolution?

[D/L-Asp + 2α + Na + K]2+

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3-amino 3-deoxy α-cyclodextrin (α)

D-aspartic acid L-aspartic acid

2 sec on-board accumulation followed by 4.5 m separation

Broad initial ion packet

WITHOUT CRIMPExperimental step # Passes Total path length (m)2 sec on-board accumulation 0 4.5Separation 1 18Separation 4 58.5

WITH CRIMPExperimental step # Passes Total path length (m)2 sec on-board accumulation 0 4.5CRIMP 1 18Separation 4 58.5

Effect of CRIMP on Eventual Resolution?

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[D/L-Asp + 2α + Na + K]2+

Effect of CRIMP on Eventual Resolution?

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WITHOUT CRIMPExperimental step # Passes Total path length (m)2 sec on-board accumulation 0 4.5Separation 1 18Separation 4 58.5

WITH CRIMPExperimental step # Passes Total path length (m)2 sec on-board accumulation 0 4.5CRIMP 1 18Separation 4 58.5

[D/L-Asp + 2α + Na + K]2+

Benefit of Initial CRIMP Step for Eventual Resolution

Increased sensitivity from accumulation of large ion population (2 sec)

Overcome initially broad ion distribution by compression of initial ion packet (via CRIMP) prior to long path separation results in higher resolution than when no initial CRIMP step is performed 21

− D-Asp− L-Asp

Will this same approach hold up for other D/L pairs?

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− D-Ser

[Ser + 2α + Li + H + H2O]2+

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− D/L-Sermixture

− L-Ser

58.5 meter separation with 2 sec on-board accumulation and initial CRIMP step

− D/L-Thr mixture

− D-Thr

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[Thr + 2α + Li + H + H2O]2+

− L-Thr


What effect does an increase in cyclodextrin cavity size (alpha vs. beta) have on enantiomeric separations?

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3-amino 3-deoxy α-cyclodextrin (α) 3-amino 3-deoxy β-cyclodextrin (β)

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[Asp + 2CD + K + H]2+


Broader peaks and noisier signal for β-CD complexes versus α-CD ones

− D-Asp− L-Asp − L-Asp − D-Asp

α-CD β-CD

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[Ser + 2CD + Li + H + H2O]2+


Poorer resolution as compared to when α-CD was used for D/L-Ser

− L-Ser− D-Ser − L-Ser− D-Ser

α-CD β-CD

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[Thr + 2CD + Li + H + H2O]2+


Poorer resolution as compared to when α-CD was used for D/L-Thr

α-CD β-CD

− D-Thr − L-Thr − D-Thr − L-Thr

Could other isomeric analytes be separated through this cyclodextrin-based complexation strategy?

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72 meter separation of bile acid mixture with on-board accumulation and CRIMP step

Bile Acid Isomer Separations

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[M + K]+

[M − H]−

72 meter separation of bile acid mixture with on-board accumulation and CRIMP step[M + α + H + K]2+ with peak assignments based on individual runs

α-Cyclodextrin-Based Bile Acid Isomer Separations

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TUDCA

TCDCA THDCA

TDCA

72 meter separation of bile acid mixture with on-board accumulation and CRIMP step[M + α + H + K]2+ with peak assignments based on individual runs

Larger analyte (bile acid) causes inclusion complex to be a 1:1 ratio (CD:BA) rather than 2:1 as observed for smaller amino acids 32

α-Cyclodextrin-Based Bile Acid Isomer Separations

GCDCAGUDCA

GDCA

Conclusions

Cyclodextrins are suitable chiral modifiers and have potential for broader utility in IM-based enantioseparations

α-cyclodextrin-based complexes provides higher resolution than β-complexes for both amino acid enantiomers and bile acid isomers

SLIM IM-MS separation does not perturb or fragment these non-covalent complexes

SLIM IM-MS enables higher sensitivity and higher resolution enantiomeric separations compared to existing IM-MS platforms

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AcknowledgementsSoftware/HardwareSpencer Prost Randy NorheimGordon Anderson

SLIM TeamRichard D. SmithErin BakerYehia IbrahimChris ChouinardIan WebbSandilya GarimellaRoza WojcikAilin LiKwame AttahAneesh Prabhakaran

FundingDOE Office of Biological and Environmental Research

NIGMS P41 Biomedical Technology Proteomics Research ResourceMOBILion Systems, Inc.

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high-resolution enantiomeric separations on a slim im-ms … · 2018. 7. 16. · traveling wave ion...

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