Differential Mobility Spectrometry with the AB SCIEX SelexION™ Technology for 5500 Systems A New Dimension of Selectivity Mauro Aiello, Senior Product Manager

2 © 2011 AB SCIEX

AB SCIEX SelexION™ Technology

Differential Mobility Spectrometry with the SelexION™ Technology

for 5500 Systems delivers a new dimension of selectivity and

performance for any application requiring the separation of isobaric

species, isolation of challenging co-eluting contaminants and reduction

of high background noise.

3 © 2011 AB SCIEX

AB SCIEX SelexION™ Technology

• Robust hardware designed for ease of use

•Few minutes to install/remove

•No tools required

•No need to break vacuum

4 © 2011 AB SCIEX

QTRAP® 5500 System Ion Path with SelexION™ Technology

SelexION™ Technology ion path components

5 © 2011 AB SCIEX

How does SelexION™ Technology separate Ions?

Separation waveform (SV):

Radially displaces ions towards

one or the other electrode,

depending upon high and low

field mobility characteristics

Compensation voltage (COV):

Restores the trajectory for a

given ion to allow them to

transmit through the DMS device

and enter the mass

spectrometer

SV COV

To

MS Gas

flow

•Differential Mobility Spectrometry (DMS) is the term used for planar geometry

6 © 2011 AB SCIEX

Advantages of Planar DMS geometry

Short residence times

Rapid voltage changes for MRM operation

– MRM cycle times of 25 msec, (20 msec pause time)

– Fast LC support

Transparent Mode

– Allows all ions to be transmitted by turning off voltages

Minimal diffusion losses

Homogeneous electric fields within the DMS analyzer.

– Improved resolution at high voltages

Uniform conditions for the addition of chemical modifiers

– More on this to come…

Also:

– Scheduled MRM™ support

7 © 2011 AB SCIEX

Modified Transport Gases Improve Separations

•Liquid modifiers can be added to the curtain gas flow

•Improves separations

•More options for separation in difficult cases

11 compounds: methylhistamine, minoxidil, ephedrine, norfentanyl,

acyclovir, clenbuterol, tramadol, quinoxifen, pamaquin, fendiline,

buscopan.

1.0

0.8

0.6

0.4

0.2

0.0

-60 -40 -20 0

1.0

0.8

0.6

0.4

0.2

0.0

Norm

aliz

ed S

ignal

-60 -40 -20 0

CV (V)

Nitrogen Transport Gas

118 Td

7x improved separation

1.5% IPA

8 © 2011 AB SCIEX

Separations are Chemical in Nature

High field

Declustering

Mobility Increases

Low Field

Clustering-

Mobility Decreases

+

Krylov et al., Int. J. Mass Spectrom., 2009, 285, 149-156

High field

Declustering

Mobility Increases

Low Field

Clustering-

Mobility Decreases

Dynamic Cluster/Decluster Model

9 © 2011 AB SCIEX

Effects of Different Modifiers Gas phase LC?

1.0

0.8

0.6

0.4

0.2

0.0

Norm

aliz

ed S

ignal

-50 -40 -30 -20 -10 0

CV (V)

Isopropanol Separation

1.0

0.8

0.6

0.4

0.2

0.0

Norm

aliz

ed S

ignal

-50 -40 -30 -20 -10 0

CV (V)

Acetone Separation

1.0

0.8

0.6

0.4

0.2

0.0

Norm

aliz

ed S

ignal

-50 -40 -30 -20 -10 0

CV (V)

Acetonitrile Separation

Red: Ephedrine

Black: Acyclovir

Green: Norfentanyl

Blue: Clenbuterol

Purple: Imipramine

Brown: Diazepam

Pink: Quinoxifen

10 © 2011 AB SCIEX

Clenbuterol analysis from urine samples

LC-MSMS analysis of clenbuterol from urine samples is known to exhibit high degree of interferences in all major MRM transitions monitored.

These interferences also vary greatly in terms of complexity and intensity levels between subject samples.

Here we investigated the use of DMS to increase the selectivity of the LC-MSMS analysis

11 © 2011 AB SCIEX

Clenbuterol Spiked in Human Urine (dil. 1:1 prior to analysis) QTRAP® 5500 vs 5500 with SelexION™ Technology

MRM

276-203

MRM

276-168

MRM

276-132 QTRAP

5500

SelexION

Technology

on 5500

12 © 2011 AB SCIEX

Eliminating High Chemical Noise with SelexION™ Technology

Pentoxifylline ~20 times LOQ Improvement

10 pg/uL

QTRAP® 5500 With SelexION

QTRAP® 5500

13 © 2011 AB SCIEX

Eliminating High Chemical Noise with SelexION™ Technology

Pentoxifylline ~20 times LOQ Improvement

1 pg/uL

QTRAP® 5500 With SelexION

QTRAP® 5500

14 © 2011 AB SCIEX

Measuring Large Peptides BNP +6 Charge State

For some large endogenous peptides that don’t fragment well, monitoring intact by SIM can provide good sensitivity but is prone to interferences

DMS can significantly reduce the presence of a co-eluting nearly isobaric matrix peak, to improve detection limits

3.2 3.4 3.6 3.8

Time, min

0

2e4

4e4

Inte

nsity

Blank – DMS off

Blank – DMS on

128 pg/mL

640 pg/mL

15 © 2011 AB SCIEX

N

NNH

N

NN

O

OH

N

NN

OH

OHO

Regular QTRAP® 5500

With SelexION™ technology

MRM

70-43

MRM

158-70

MRM

128-70

1.0 2.0 3.0 Time, min

1.0 2.0 3.0 Time, min

1.705

Triazole other pesticide degradants in Grapefruit

Grape fruit extract fortified with conazole degradant products at 0.01mg/kg. Samples kindly supplied by R. Schoening, Bayer CropScience, Monheim, Germany, & J. Jasak,

Technical University, Institute of Food Chemistry, Dresden, Germany

Triazole acetic acid Triazole lactic acid Triazole

1.0 2.0 3.0 Time, min

1.714

16 © 2011 AB SCIEX

Peak area Buprenorphine, CV% = 6.8%

Peak area Clenbuterol, CV% = 5.6%

IS corrected Peak area Buprenorphine, CV% = 1.7%

IS corrected Peak area Clenbuterol, CV% = 1.6%

QTRAP® 5500 System with SelexION™ Technology

Robustness for Demanding Applications in regulated labs

1000 plasma samples injected over 66 hours

17 © 2011 AB SCIEX

Separation of Isobaric Compounds (m/z 309)

1.0

0.8

0.6

0.4

0.2

0.0

No

rma

lize

d S

ign

al

-50 -40 -30 -20 -10 0

CV (V)

Bestatin

Warfarin Phenylbutazone

Quinoxyfen

Benoxinate

SelexION™ Technology Applications

18 © 2011 AB SCIEX

Separation of Isomers - Pseudoephedrine/Ephedrine

Pseudoephedrine Ephedrine

•Separation of pseudoephedrine and ephedrine with

SelexION™

•Indistinguishable by MS or MS/MS

SelexION™ Technology Applications

19 © 2011 AB SCIEX

Steroid Interference Removal with SelexION™ Technology

20 © 2011 AB SCIEX

Experiment Conditions

5 subjects each of male and female serum samples obtained from a customer laboratory

STD was prepared in pooled children under age 7 serum samples.

Two sample preparation methods:

– LLE with 90/10 hexane/ ethyl acetate ( 200 uL serum; 1 mL solvent; recon 150 uL with 50/50 MeOH/H2O)

– PPT with ACN (200 uL serum; 600 ACN; dry down and recon 150 uL with 50/50 MeOH/H2O)

Column: Kinetex 2.6 u 50x2.1

mm; run time: 7 min; flow rate

0.3 mL/min

MPA: Water+0.1% FA;

MPB: MeOH+0.1% FA

ESI: positive

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Testosterone Female #3 Serum Sample: LLE

DMS OFF/

289/109 DMS OFF/

289/97

DMS ON/

289/109 DMS ON/

289/97

Interference

Ion ratio~21 Peak of interested Interference

Peak of interested

Ion Ratio=1.28

22 © 2011 AB SCIEX

Testosterone Female #3 Serum Sample: PPT

DMS OFF/

289/109

DMS OFF/

289/97

DMS ON/

289/109

DMS ON/

289/97

Peak of interested Peak of interested

@ RT 3.37 min

23 © 2011 AB SCIEX

STD Curve in Serum Sample with DMS on (PPT)

4 female

subjects

Sample Neat (n=28)

Serum Sample

PPT (n=25)

Serum Sample

LLE (n=25)

Average Ion Ratio

(97/109) 1.23 1.28 1.24

STDEV 0.04 0.07 0.04

%CV 3.44 5.47 3.12

Accuracy 96- 105%

24 © 2011 AB SCIEX

Steroids panel: androstenedione at RT=8.14 min / protein precipitation sample preparation

DMS OFF MRM 287/109

DMS OFF MRM 287/97

Peak of interest

Peak of interest

Peak of interest

Peak of interest

DMS ON MRM 287/109

DMS ON MRM 287/97

25 © 2011 AB SCIEX

DMS as separation technique when LC is not an option…

Infusion based analysis

Surface Sampling

– LESA

Direct desorption technique

– DESI

– LDTD

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Advion LESA (Liquid Extraction Surface Analysis)

27 © 2011 AB SCIEX

Propranolol Glucuronide Structural

Isomers (acyl glucuronide)

Aromatic Glucuronide

Aliphatic Glucuronide

Isobaric metabolites

Same fragments and dominant MRM transition!

Direct Tissue Profiling using the Advion LESA instrument

DMS separation with 1.5% ACN modifier

DMS resolves propranolol and the 2

glucuronide structural isomers!

CV = -17.4 V

CV = -10.4 V

28 © 2011 AB SCIEX

LDTD with SelexION™ Technology

LDTD on 5500 with DMS

22X background

reduction

2576 cps

J Wu et al., Anal. Chem., 2007, 79, 4657-4665.

2.5 pg Clozapine in protein precipitated plasma

LDTD on 5500

5576 cps

Hesham Ghobarah, Yves Leblanc, Michael Jarvis, Adrian Taylor, and Pierre Picard

Thermal desorption/APCI

Low

STD

Matrix

BLANK

30 © 2011 AB SCIEX

Summary

An added dimension of selectivity through differential mobility spectrometry with the AB SCIEX SelexION™ Technology on the QTRAP® 5500 system and the Triple Quad™ 5500 system

Improvement in data quality and LOQs for assays with interferences

Robust and reliable for use in a regulated environment

Improved throughput – Less need for chromatographic separations and sample prep

– Can be coupled to non-LC sample introduction

Combined with MRM3 on the QTRAP 5500 system, SelexION Technology gives several options

– High sensitivity in MRM mode

– High selectivity for analytes that fragment well with MRM3

– High selectivity for analytes that fragment poorly with DMS-MS or DMS-MS/MS with the SelexION Technology

– Separation for isobaric analytes with common fragments

31 © 2011 AB SCIEX

Thank You

32 © 2011 AB SCIEX

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© 2011 AB SCIEX. All rights reserved. Information subject to change without notice.