Download pdf - Come spingere al limite le misure degli ormoni steroidei ... · 1 Come spingere al limite le misure degli ormoni steroidei per LC-MS/MS. Bruno Casetta AB Sciex Consultant Clinical

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Come spingere al limite le misure degli ormoni steroidei per LC-MS/MS.

Bruno CasettaAB Sciex Consultant Clinical Applications Mass Spectrometry.

2 © 2011 AB SCIEX

2

3 © 2011 AB SCIEX

Il problema della accuratezza degli IAs

Lack o f S pec ific ity o f Im m unoassaysD ata from C A P P T P rog ram

An a lytes Low H igh

17 O H P rogeste rone(ng /dL) 16.0 82.7 (5 .2 )

Estrad io l (pg /m L) 12.3 210.7 (17.1 )

P rogeste rone (ng /m L) 7 .90 23.9 (3 .0)

4 © 2011 AB SCIEX

Il pattern metabolico degli steroidi

3

5 © 2011 AB SCIEX

From:From:From:From: Jean M. Lacey, Carla Z. Minutti, Mark J. Magera, Angela L. Tauscher, Bruno Casetta, Mark McCann, James Lymp, Si Houn Hahn, Piero Rinaldo,and Dietrich Matern, Clin Chem 2004; 50: 621Clin Chem 2004; 50: 621Clin Chem 2004; 50: 621Clin Chem 2004; 50: 621----625625625625

Steroid profiling by MS/MS in blood spots

CortisolCortisolCortisolCortisol

D4D4D4D4----AndrostenedioneAndrostenedioneAndrostenedioneAndrostenedione

d8d8d8d8---- 17OH17OH17OH17OH----Progesterone (IS)Progesterone (IS)Progesterone (IS)Progesterone (IS)

17OH17OH17OH17OH----ProgesteroneProgesteroneProgesteroneProgesteroneControlControlControlControl

False positive for CAHFalse positive for CAHFalse positive for CAHFalse positive for CAH

CAHCAHCAHCAH

6 © 2011 AB SCIEX

AB SCIEX TOF/TOF™Systems

iTRAQ®

reagents

QTRAP® 5500 System

4000 QTRAP®

System

mTRAQ™ reagents

Scheduled MRM™ Algorithm

AB SCIEX TripleTOF™5600 System

???

4

7 © 2011 AB SCIEX

Q0 Q1 Q2 Q3

LIT

Q0 Q1 Q2 Q3Q0 Q1 Q2 Q3

FragmentionMol.Ion Fragm.Ions

Il Triplo Quadrupolo

8 © 2011 AB SCIEX

Stable and unstable Ion trajectories

At any RF/DC potential setting only ions of one particular mass/charge ratio have a stable trajectory

5

9 © 2011 AB SCIEX

Pass Precursor Check for markerCAD

Q1 LINAC Q2 Q3

Signal after Q3

m/z

Ionized Analyte Specific fragment

Multiple Reaction Monitoring

Nel Quadrupolo in SIM o SRM, il Duty Cycle è vicino

al 100%: tutti gli ioni prodotti dalla sorgente sono

utilizzati per acquisire la misura.

10 © 2011 AB SCIEX

The workflow for measurements with MSMS

Affordable Sample Prep LC desires/requ.

LC/MS/MS instrument with maximum Sensitivity and Robustness

BLOOD

Specimen Amount

6

11 © 2011 AB SCIEX

17-OHP performances

Kushnir 2006b Serum/Plasma SPE, NH2OH, LLE API 4000-TIS-pos 200 uL 40 pg/mL 50 pg 2Ceglarek 2008 Serum PPT, 2D API 4000-APCI-pos 100 uL 33 pg/mL 100 pg 3.3Lacey 2004 DBS-4.8mm LLE API 3000-TIS-pos 10 uL 4 pg/mL 1900 pg 7.6Guo 2006 Serum PPT, false2D API 5000-APPI-pos 200 uL 135 pg/mL 1.5 pg 0.2025Rauh 2006 Serum/Plasma/saliva PPT, 2D API 4000-APCI-pos 100 uL 20 pg/mL 100 pg 2Janzen 2008 Serum/DBS PPT, dry W Ultima-ESI-pos 15 uL 12 pg/mL 325 pg 3.9Fanelli 2010 Serum PPT,off-line SPE,2D API 4000-APCI-pos 900 uL 600 pg/mL 9.8 pg 1.7iMethod Serum PPT, 1D diverted API 5X00-APCI-pos 200 uL 33 pg/mL 100 pg 3.3PE-Kit Serum PPT, dry API 5000-APCI-pos 100 uL 80 pg/mL 38 pg 3.04

LOQ

12 © 2011 AB SCIEX


Instrument

17-OHP performances

7

13 © 2011 AB SCIEX


Sample preparation

17-OHP performances

14 © 2011 AB SCIEX


Starting Sample Volume Sample Volume

consumed

17-OHP performances

On-column pg

for LOQ

8

15 © 2011 AB SCIEX

A Steroids commercial KitXIC of -MRM (4 pairs): Period 1, 359.200/189.100 Da ID: ALDO 1 from Sample 2 (Well 2-CoLOW) of 0307-Plate1-row3.wiff (Heated Nebulizer), ... Max. 1193.4 cps.

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 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 12.5 13.0Time, min

0.0

1.0e4

2.0e4

3.0e4

4.0e4

5.0e4

6.0e4

7.0e4

8.0e4

9.0e4

1.0e5

1.1e5

1.2e5

1.3e5

1.4e5

1.5e5

1.6e5

1.7e5

1.8e5

1.9e5

2.0e5

2.1e5

2.2e5

Inte

nsity

, cps

x 20.0

ALDO

CORT

DHEAS

CCONE

11-DECOL

ADIONE

TESTO

17-OHP

DHEA

PROG

16 © 2011 AB SCIEX

Corticosteroids Positive Ion Product Ion Spectra

•Corticosterone

•11-Desoxycortisol

•21-Desoxycortisol

+ M S2 (3 47.3 0): 20 M CA sca ns from Sa mple 1 (21 Desoxy Pos) o f AP CI P ro duct Ion 102604.w iff (Hea ted Ne bulizer) Ma x. 2.5e 5 cps .

60 80 10 0 12 0 140 160 180 20 0 2 20 2 40 260 280 300 32 0 340 3 60 380 400m/z, am u

2.0e 4

4.0e 4

6.0e 4

8.0e 4

1.0e 5

1.2e 5

1.4e 5

1.6e 5

1.8e 5

2.0e 5

2.2e 5

2.4e 52.5e 5 1 21.3

97.2

11 9.3 123.3147.2

10 5.1

119.0131.3 169.2

135.2 173 .393.2 163.1161.4

181 .3129.0 155.1 209.3185.1141.383.179.055.3 183.1 225.487 .0 207.2149.5111.259.3 199 .576.7 239 .361.3 253 .3232 .4 260.3 296.1 300 .3 318.1

+ M S2 (347.50): 20 MC A scans from S am ple 3 (C omp ound B pos) of A PCI Product Ion 1026 04.w iff (H eated N ebulize r) Ma x. 1 .9 e6 cp s.

60 80 100 12 0 140 1 60 180 200 220 2 40 260 280 30 0 320 340 360m/z, am u

2.0 e5

4.0 e5

6.0 e5

8.0 e5

1.0 e6

1.2 e6

1.4 e6

1.6 e6

1.8 e6

1.9 e61 21.2

97.3

105.11 23.2

1 09.2 14 5.293.0

133.195.3 159.1 16 3.28 3.27 9.1 135 .31 17.1

169.3161.267.2 173 .2128.2 1 97.2149.287.3 116 .8 167.3 2 11.2101.4 154.276.8 223 .4 227 .27 1.257.1 192.1 251.3 255.3 271.2 299.3 347.0

+M S2 (347.30): 20 M CA scans from S am ple 5 (Com pound S) o f APC I Product Ion 102604.w iff (Heated N ebulizer) M ax. 3 .1e6 cps.

60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360m /z, am u

2.0e5

4.0e5

6.0e5

8.0e5

1.0e6

1.2e6

1.4e6

1.6e6

1.8e6

2.0e6

2.2e6

2.4e6

2.6e6

2.8e6

3.0e6

109.2

97.1

123.293.181.3 105.1 145.3 159.2131.1 169.31 17.169.3 185.255.0 197.2 211.1 225.4 237.3 253.676.7 269.399.1 293.5

9

17 © 2011 AB SCIEX

1 2 3 4 5 6 7Time, min

0.0

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

21 DESOX

CORTICOSTERONE

11 DESOX

17 OH PREGNENOLONE

ANDROSTANEDIOL

Isobaric SteroidsSeparation

LC separation, why do we need it?

18 © 2011 AB SCIEX

Sample preparation for Steroids measurements with MSMS

Just Sampledilution

Off-line SPE

LLE

PPT and On-linepurification

10

19 © 2011 AB SCIEX

Multiplex FIA - LC/MS/MS for NBS-CAH

AB Sciex 5500 System withTurboV TIS Source

Autosampler

Pump (LC)

Pump (FIA)

20 © 2011 AB SCIEX

The sequenceFIA - LC/MS/MS

FIA (NBS)

Autosampler

LC (CAH)

FIA (NBS)

11

21 © 2011 AB SCIEX

Principle of multiplexing NBS and CAH

XIC of +MRM (5 pairs): Period 2, 363.3/121.1 Da ID: Cortisol from Sample 3 (Mix80nM-Phe1uM) of ag03-Fia8020-a0_3b1_1unWashed.wiff (Turbo... Max. 3.1e4 cps.

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8Time, min

0.0

5.0e4

1.0e5

1.5e5

2.0e5

2.5e5

3.0e5

3.5e5

4.0e5

4.5e5

5.0e5

5.5e5

6.0e5

6.5e5

6.9e5

x 5.0

1.20

22 © 2011 AB SCIEX

XIC of +MRM (5 pairs): Period 2, 363.3/121.1 Da ID: Cortisol from Sample 14 (Sample5-Vial2) of ag06-Protocol.wiff (Turbo Spray), Smoothed, S... Max. 8693.8 cps.

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0Time, min

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

x 8.0

1.38

+NL (46.00): Period 1, Exp 1, 0.201 to 1.003 min from Sample 14 (Sample5-Vial2) of ag06-Protocol.wiff (Turbo Spray) Max. 5.3e5 cps.

70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230m/z, Da

0.0

5.0e4

1.0e5

1.5e5

2.0e5

2.5e5

3.0e5

3.5e5

4.0e5

4.5e5

5.0e5

5.3e5132.2

118.2

166.2

116.2

104.2170.1

90.0156.1

133.1

102.7 182.0130.1

Underiv. AA and AC

Cortisol

21-deoxycortisol

11-deoxycortisol

Androstenedione

17-OH-Progest.

12

23 © 2011 AB SCIEX

Comparison with performances retreived from published papers.

ANALYTE Reference Matrix Strategy Instrument

Starting Specimen Volume

Injected Specimen

Volume uLLOQ units value

On column units value

Progestagens (21C)

17-Hydroxyprogesterone (17OHP) Kushnir 2006b Serum/Plasma SPE, NH2OH, LLE API 4000-TIS-pos 200 uL 40 pg/mL 50 pg 217-Hydroxyprogesterone (17OHP) Ceglarek 2008 Serum PPT, 2D API 4000-APCI-pos 100 uL 33 pg/mL 100 pg 3.317-Hydroxyprogesterone (17OHP) Lacey 2004 DBS-4.8mm LLE API 3000-TIS-pos 10 uL 4 pg/mL 1900 pg 7.617-Hydroxyprogesterone (17OHP) Guo 2006 Serum PPT, false2D API 5000-APPI-pos 200 uL 135 pg/mL 1.5 pg 0.202517-Hydroxyprogesterone (17OHP) Rauh 2006 Serum/Plasma/Saliva PPT, 2D API 4000-APCI-pos 100 uL 20 pg/mL 100 pg 217-Hydroxyprogesterone (17OHP) NBS-CAH DBS-5mm Extr. DBS, Mpx API 5500-TIS-pos 12 uL 1 pg/mL 2000 pg 2

Vitamin D analysis by LC/MS/MS

13

25 © 2011 AB SCIEX

25(OH)D Cholecalciferol(Vitamin D3)

7-dehydro-cholesterol

KIDNEY 1,25(OH)2Vitamin D3

is the Calcium regulating hormone

1000 x LOWER then 25(OH)vitamin D3

UVB LIGHT

LIVER

25(OH)D3Cholecalciferol(Vitamin D3)

7-dehydro-cholesterol

LIVER

UVB

25(OH)D is the accepted measure of vitamin D nutritional status

26 © 2011 AB SCIEX

Calcifediol

Calcitriol

Colecalciferol

14

27 © 2011 AB SCIEX

Classical RIA methodology performances

� Imprecision: 10 % CV to 15 % CV (between run)

� Sensitivity: 24 pmol/L (10 pg/mL)

� Dynamic range: 24-600 pmol/L (10-250 pg/mL)

� Reference range: 48-192 pmol/L (20-80 pg/mL)

� Sample volume: 1.5 mL serum RIA liquid scintillation counting (tritium)

UZ Leuven Gasthuisberg Belgium

28 © 2011 AB SCIEX

The hardware configuration as depicted in the schematic includes:

- an AB SCIEX Triple Quad™ 5500 Mass Spectrometer equipped with a Turbo V source and the Electrospray probe, and with

- an external 10 port-2 way Valco-valve

- an LC-stack including: - 2 binary pumps- autosampler

- a Perfusion POROS® column R1/10 4.6 x 50 mm.

- two Onyx columns 3 x 100 mm

MATERIALS

15

29 © 2011 AB SCIEX

AB SCIEX Triple Quad 5500 MASS SPECTROMETER

WITH ESI SOURCEAutosampler

SEPARATION Pump

LOADING Pump

1,25(OH)2-Vitamin D 2/3 (and optionally 25(OH)-Vitamin D2/3) measurement with Sample On-Line Cleanup.

Waste

POROS column

Monolithic columns

H2O / ACN-MetOH MixH2O / MetOH + Li +

30 © 2011 AB SCIEX

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0Time, min

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

5500

6000

6500

7000

75007728 15.41

24(R),25(OH)2-Vit.D3

25(S),26(OH)2-Vit.D3

1α,25(OH)2-Vit.D3

d6-1α,25(OH)2-Vit.D3

14.5 14.6 14.7 14.8 14.9 15.0 15.1 15.2 15.3 15.4 15.5 15.6 15.7 15.8 15.9 16.0 16.1 16.2 16.3 16.4Time, min

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

5500

6000

6500

7000

75007728

15.41

The Di-Hydroxy-Vit.D 3 isomers separation

16

31 © 2011 AB SCIEX

Data from external Lab.

Imprecision (n=11 between day)

Concentration (pg/ml)

%CV

11.1 14.3

17.1 13.7

23.6 13.6

50.5 8.6

79.6 4


Stability of the medium control

(120 pmol/l – 50 pg/ml)


Start of series Mid series End of series

16-10-09 49.6 55.5 57.4

19-10-09 50.5 53.0 50.6

26-10-09 52.5 55.5 54.6

29-10-09 46.5 51.3 46.7

9-11-09 54.9 52.8 54.4

12-11-09 47.6 56.6 44.0

19-11-0948.4 48.4 45.1 48.5

Method comparison n=76


32 © 2011 AB SCIEX

A reduced sample size of only 30 uL of plasma is consumed for the reliablequantitation of calcitriol

The LOQ achievable is < 10 pg/mL (< 24 pmol/L),

The LOD is ~ 3 pg/mL (< 7 pmol/L)

The intra-run precisions have been < 20 % @ 20 pg/mL (48 pmol/L) and better than 5 % at 100 pg/mL (240 pmol/L).

Bruno CASETTA, Ivo JANS, Jaak BILLEN, Dirk VANDERSCHUEREN and Roger BOUILLON European Journal of Mass Spectrometry, Volume 16, 81-89 (2009).

Performance keypoints

17

Serum E1 and E2.

34 © 2011 AB SCIEX

A first methodology for the E1 and E2.

18

35 © 2011 AB SCIEX

The 5 pg/mL-spiked serum sample ......

36 © 2011 AB SCIEX

AB SCIEX TRIPLE QUADRUPOLE MASS SPECTROMETER

WITH ESI SOURCEAutosampler

SEPARATION Pump

LOADING Pump

The approach with a “2D” Sample Focusing Injection and On-Line Cleanup for LLE.

Waste

Trapping column

H2O (A) / MetOH-ACN (B)H2O + F- / MetOH

B

A

Built-in Valve

Analytical column

19

37 © 2011 AB SCIEX

Low serum sample XIC chromatogram as integrated by MultiQuant software for E2

38 © 2011 AB SCIEX

Calibration with “New Method” on E2-spiked serum samples

20

39 © 2011 AB SCIEX

Correlation “New Method” with IA

y = 0.7464x + 0.7998

R2 = 0.9991

0

50

100

150

200

250

300

0 100 200 300 400

pg/mL IA

pg/m

L LC

-MS

MS

Series1

Linear (Series1)

40 © 2011 AB SCIEX

Sample Prep.

1 Vol. Serum 2 Vol. ACN

21

41 © 2011 AB SCIEX

Endogenous E1, E2 in Plasma by PPT and 2D-approach, Option 1

XIC of -MRM (4 pairs): 269.100/145.100 Da ID: E1 from... Max. 3.1e6 cps.

0 2 4 6 8 10 12Time, min

0.0

2.0e5

4.0e5

6.0e5

8.0e5

1.0e6

1.2e6

1.4e6

1.6e6

1.8e6

2.0e6

2.2e6

2.4e6

2.6e6

2.8e6

3.0e6

3.1e6

Intensity, cps

10.29

XIC of -MRM (4 pairs): 273.100/147.100 Da ID: d4E1 f... Max. 1412.5 cps.

0 2 4 6 8 10 12Time, min

0

1000

2000

3000

4000

5000

In

te

ns

ity

,

cp

s

XIC of -MRM (4 pairs): 269.100/145.100 Da ID: E1 fro... Max. 3595.8 cps.

0 2 4 6 8 10 12Time, min

200

400

600

800

1000

In

te

ns

ity

,

cp

s


0 2 4 6 8 10 12Time, min

200

400

600

800

1000I

nt

en

sit

y,

c

ps

PlasmaE1

E2

XIC of -MRM (4 pairs): 269.100/145.100 Da ID: E1 from Sample 2 (Ex 25 ppb) of 111014-250pg-C8.wiff (Turbo Spray) Max. 3.1e6 cps.

9.3 9.4 9.5 9.6 9.7 9.8 9.9 10.0 10.1 10.2 10.3 10.4 10.5 10.6 10.7 10.8 10.9 11.0 11.1 11.2 11.3 11.4Time, min

0.0

2.0e5

4.0e5

6.0e5

8.0e5

1.0e6

1.2e6

1.4e6

1.6e6

1.8e6

2.0e6

2.2e6

2.4e6

2.6e6

2.8e6

3.0e6

3.1e6 10.29

E2

E1

XIC of -MRM (4 pairs): 273.100/147.100 Da ID: d4E1 from Sample 2 (PlasmaACN) of 111014-Plasma-C8.wiff (Turbo Spray), Smoothe... Max. 1412.5 cps.

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 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 12.5Time, min

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

XIC of -MRM (4 pairs): 269.100/145.100 Da ID: E1 from Sample 2 (PlasmaACN) of 111014-Plasma-C8.wiff (Turbo Spray), Smoothed, ... Max. 3595.8 cps.

6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0Time, min

100

200

300

400

500

600

700

800

900

1000

1100

1200

1300

E1

XIC of -MRM (4 pairs): 271.100/145.100 Da ID: E2 from Sample 2 (PlasmaACN) of 111014-Plasma-C8.wiff (Turbo Spray), Smoothed, ... Max. 1437.8 cps.

6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0Time, min

100

200

300

400

500

600

700

800

900

1000

1100

1200

1300

10.48

E2

42 © 2011 AB SCIEX

Endogenous E1, E2 in Plasma by PPT and 2D-approach, Option 2

XIC of -MRM (4 pairs): 269.100/145.100 Da ID: E1 from... Max. 1.1e6 cps.

0 2 4 6 8 10 12Time, min

0.00

5.00e4

1.00e5

1.50e5

2.00e5

2.50e5

3.00e5

3.50e5

4.00e5

4.50e5

5.00e5

5.50e5

6.00e5

6.50e5

7.00e5

7.50e5

8.00e5

8.50e5

9.00e5

9.50e5

1.00e6

1.05e61.08e6

Intensity, cps

11.02


0 2 4 6 8 10 12Time, min

2000.00

4000.00

6000.00

8000.00

1.00e4

In

te

ns

ity

,

cp

s


0 2 4 6 8 10 12Time, min

0

2000

4000

6000

In

te

ns

ity

,

cp

s

11.01


0 2 4 6 8 10 12Time, min

0

1000

2000

3000

3480

In

te

ns

ity

,

cp

s

4.42

11.623.15

5.91 11.056.12

E1

E2

Plasma

XIC of -MRM (4 pairs): 269.100/145.100 Da ID: E1 from Sample 4 (Ex 25 ppb) of 111017-50pg-PF35t90i6.wiff (Turbo Spray), Smoothe... Max. 9.5e5 cps.

10.3 10.4 10.5 10.6 10.7 10.8 10.9 11.0 11.1 11.2 11.3 11.4 11.5 11.6 11.7 11.8 11.9 12.0 12.1 12.2 12.3 12.4 12.5 12.6Time, min

0.0

5.0e4

1.0e5

1.5e5

2.0e5

2.5e5

3.0e5

3.5e5

4.0e5

4.5e5

5.0e5

5.5e5

6.0e5

6.5e5

7.0e5

7.5e5

8.0e5

8.5e5

9.0e5

9.5e511.02

E1

E2

XIC of -MRM (4 pairs): 275.100/147.100 Da ID: d4E2 from Sample 2 (PlasmaACN) of 111017-Plasma-PF35t90i6.wiff (Turbo Spray), S... Max. 4.5e4 cps.

6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0 12.5Time, min

0.00

500.00

1000.00

1500.00

2000.00

2500.00

3000.00

3500.00

4000.00

4500.00

5000.00

5500.00

6000.00

6500.00

7000.00

7500.00

8000.00

8500.00

9000.00

9500.00

1.00e4

1.05e4

1.10e4

E1

E2

XIC of -MRM (4 pairs): 271.100/145.100 Da ID: E2 from Sample 2 (PlasmaACN) of 111017-Plasma-PF35t90i6.wiff (Turbo Spray), Sm... Max. 3480.4 cps.

7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0 12.5Time, min

0

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

2400

2600

2800

E2

22

SelexION™ Ion Mobility Technology:Introducing a New Dimension of Selectivity for LC-MS/MS Analysis

44 © 2011 AB SCIEX

SelexION™ Ion Mobility Device� Separates analyte ions based upon differences in their low-field and high-field

mobilities.

� The application of alternating low and high electric fields in the region between two planar electrodes causes analyte ions to migrate towards one of the electrodes.

� Ions are steered back towards the center-line using a compound-specific DC compensation voltage.

New hardware

23

45 © 2011 AB SCIEX

SelexION™ Ion Mobility Device

to mass spectrometer

Compensation Voltage (COV)

ions in transport gas

to mass spectrometer

Compensation Voltage (COV)

ions in transport gas

� Separates analyte ions based upon differences in their low-field and high-field mobilities.

� The application of alternating low and high electric fields in the region between two planar electrodes causes analyte ions to migrate towards one of the electrodes.

� Ions are steered back towards the center-line using a compound-specific DC compensation voltage.

46 © 2011 AB SCIEX

α>0

α<α<α<α<0

αααα=0

t1

t2

)0(

)0()()(

K

KEKE

−=α

Tuning the SelexION™ Ion Mobility Device

� Recall that ions migrate towards one of the planar electrodes– If an ion’s high-field mobility K(E) is larger than its low-field mobility K(0), α>0– If an ion’s low-field mobility K(0) is larger than it high-field mobility K(E), α<0

� Any ion can be steered back onto the center-line, by application of a compound-specific DC compensation voltage (CV).

24

47 © 2011 AB SCIEX

Tuning the SelexION™ Ion Mobility Device

� In infusion mode, the compensation voltage (CV) parameter is ramped in order to determine the optimized value.

� Note that this is analogous to tuning any other compound-specific parameter, e.g. the collision energy (CE)

-14 -12 -10 -8 -6 -4 -2 0 2 40%

50%

100%

Rel

. Int

. (%

)

Compensation Voltage (CV)-14 -12 -10 -8 -6 -4 -2 0 2 4

0%

50%

100%

Rel

. Int

. (%

)

Compensation Voltage (CV)

48 © 2011 AB SCIEX

LC/MS/MS Analysis of Prostaglandins using SelexION™ Ion Mobility Technology for Enhanced Selectivity

SelexION™ device “off”

SelexION™ device “on”COV = 10

SelexION™ device “on”COV = 7

(a)

(b)

(c)

PGF2αααα

8-iso-PGF 2αααα

PGF2αααα

8-iso-PGF 2αααα

O

OH

OH

OH

OH

CH3

PGF2α

O

OH

OH

OH

OH

CH3

PGF2α

8-iso PGF2α

O

OH

OH

OH

OH

CH3

8-iso PGF2α

O

OH

OH

OH

OH

CH3

25

49 © 2011 AB SCIEX

Optimization of the SelexION™ Ion Mobility Device

� Optimization of the SelexION™ device for isomers is accomplished by ramping the COV parameter.

� In the example shown, at COV=10, only PGF2α is transmitted. At COV=7, only 8-iso-PGF2α is transmitted.

O

OH

OH

OH

OH

CH3

PGF2α8-iso PGF2α

O

OH

OH

OH

OH

CH3

Chromatographic run-time (minutes)

50 © 2011 AB SCIEX

Testosterone Female Serum Sample: PPTEnhanced Selectivity using SelexION™Technology

DMS OFF 289/109

DMS OFF 289/97

DMS ON 289/109

DMS ON 289/97

Peak of interest Peak of interest @ RT 3.37 min

26

51 © 2011 AB SCIEX

Application examples using SelexION™ Ion Mobility Technology

52 © 2011 AB SCIEX

Enhanced separations using chemical “modifier”

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

Nor

malized

Signa

l

-60 -40 -20 0CV (V)

11 compounds: methylhistamine, minoxidil, ephedrine, norfentanyl, acyclovir, clenbuterol, tramadol, quinoxifen, pamaquin, fendiline, buscopan.

Nitrogen-only transport gas

Peak Capacity ≈ 6

1.5% isopropanol in transport gas

Peak Capacity ≈ 40

� “Modifier” is introduced by a precision liquid delivery pump

� Typical modifiers include: isopropanol, acetone, acetonitrile, methanol…

27

53 © 2011 AB SCIEX

Effects of Different Modifiers

1.0

0.8

0.6

0.4

0.2

0.0

Nor

mal

ized

Sig

nal

-50 -40 -30 -20 -10 0CV (V)

Isopropanol Separation

1.0

0.8

0.6

0.4

0.2

0.0

Nor

mal

ized

Sig

nal

-50 -40 -30 -20 -10 0CV (V)

Acetone Separation

1.0

0.8

0.6

0.4

0.2

0.0

Nor

mal

ized

Sig

nal

-50 -40 -30 -20 -10 0CV (V)

Acetonitrile Separation

Red: Ephedrine

Black: Acyclovir

Green: Norfentanyl

Blue: Clenbuterol

Purple: Imipramine

Brown: Diazepam

Pink: Quinoxifen

Thank [email protected]