1
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
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
Instrument
17-OHP performances
7
13 © 2011 AB SCIEX
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
Sample preparation
17-OHP performances
14 © 2011 AB SCIEX
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
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
UZ Leuven Gasthuisberg Belgium
Stability of the medium control
(120 pmol/l – 50 pg/ml)
UZ Leuven Gasthuisberg Belgium
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
UZ Leuven Gasthuisberg Belgium
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
XIC of -MRM (4 pairs): 271.100/145.100 Da ID: E2 fro... Max. 1437.8 cps.
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
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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
XIC of -MRM (4 pairs): 269.100/145.100 Da ID: E1 fro... Max. 7093.0 cps.
0 2 4 6 8 10 12Time, min
2000.00
4000.00
6000.00
8000.00
1.00e4
In
te
ns
ity
,
cp
s
XIC of -MRM (4 pairs): 269.100/145.100 Da ID: E1 fro... Max. 6399.9 cps.
0 2 4 6 8 10 12Time, min
0
2000
4000
6000
In
te
ns
ity
,
cp
s
11.01
XIC of -MRM (4 pairs): 271.100/145.100 Da ID: E2 fro... Max. 3480.4 cps.
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
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