View
1
Download
0
Category
Preview:
Citation preview
Method Development Challenges
• Co-eluting interferences
• High background
• Ion fragments poorly
• Long chromatographic run time
• Complicated sample preparation
Current Solutions
• Vary MS parameters– Voltages– Transitions
• Vary chromatographic conditions– Column– Mobile phase
• Revisit sample prep– Extraction condition– Derivatization
Significant time investment; no guarantee of success
FAIMS – Get there faster
Get method development results in just a few hours
Easy to Use:
– Install the FAIMS system
– Determine FAIMS condition using reference solutions
– Set FAIMS condition (1 parameter)
– Analyze your samples
Add FAIMS to your method development process
Prepare Samples
Problem with LC-MS Method
Vary MS Conditions
Use FAIMS
Vary Chromatographic
Conditions
New LC-MS Method
analyze
solved
solved
solved
not solved
not solved
not solved
Save time, save money!
FAIMS – Where does it fit?
+
++
MS Orifice Plate
Curtain Plate
Desolvation gas
+ ++
+ ++
+ ++
+
++
++ +
+
+
all types of ions transferred to the MS
FAIMS+
+
+
+++
+
+ ++
+++
+ +
+
+ ++
+ +selected ions transferred
to the MS
Curtain PlateMS Orifice Plate
Desolvation gas
th
tl
time t
volta
ge V
Vh
Vl0
y
Mobility dependent on field strength
The parallel plate FAIMS
+
Gas Flow
TypeAIon
x
th
tl
time t
volta
ge V
Vh
Vl0
y
Ion selection using a compensation voltage (CV)
The parallel plate FAIMS
-CV
+
Gas Flow
TypeAIon
x
• Operates at atmospheric pressure – between API source and MS • Uses concentric cylindrical electrodes for ion separation.• Electronic separation - voltages are used to select the ion.• Other ions are discharged to the walls of the electrodes.
+
FAIMS – How does it work?
Gas Flow
+
++
+
th
tl
time t
volta
ge V
DV
Vl
0
FAIMS - How does it help you?
Increases selectivity• In-source fragmentation• Separation of isobaric ions• Separation of positional isomers• Separation of diastereomers• Removal of endogenous interferences
• Reduces background
• Increases throughput
LC-MS/MS of a drug (10µL inj. of 5µg/mL solution) – no metabolite present in sample
Drug transition
Metabolite transition
LC-MS/MS of the N-Oxide metabolite (10µL inj. of 5µg/mL solution) - no parent drug present in sample
Problem: In-source fragmentation
• Drug and metabolite co-elute • Signal for drug is obtained even when no drug is present in sample• Will affect measured concentration of drug in sample
FAIMS-MS/MS separation of drug and metabolite
Drug m/z 488.2 → 401.1
8000
4000
0-22-17-12-7
Compensation Voltage
Metabolite m/z 504.2 → 387.0
Inte
nsit
y (c
ps)
6000
3000
0-22-17-12-7
CV = -7.2 V
CV = -18.0 V
• FAIMS separates drug and metabolite; eliminates interference formed by CID of metabolite
Effect of interference on measured drug concentration
0
10
20
30
40
50
LC-FAIMS-MS/MSLC-MS/MS
Dru
g C
onc
(ng/
mL)
Drug (25 ng/mL)
Drug (25 ng/mL) in presence of 2500 ng/mLmetabolite
• More accurate quantitation using LC-FAIMS-MS/MS
Problem: Interfering isobaric ions
Example: Pamaquin/Oxycodone/Clonazepam• Ions are isobars with similar fragment ions• Detection of these ions could be achieved by:
– Chromatographic resolution– Selection of alternate fragment ion– Different collision energy– Increased precursor ion specificity– MS3
– FAIMS
Pamaquin: Full scan single quadrupole MS
ESI-MS
ESI-FAIMS-MS
m/z 316
• ESI-MS spectrum: Many intense background ions• ESI-FAIMS-MS spectrum: Pamaquin most intense ion, background dramatically reduced
pamaquin CV = -11.5 V
oxycodone CV = -10.0 V
100
50
0
Inte
nsity
(cps
x10
-3)
-20-15-10-5Compensation Voltage
clonazepam CV = -6.0 V
FAIMS separation of pamaquin/oxycodone/clonazepam
Infusion ESI– FAIMS–MS/MS(SRM optimized for each)
• FAIMS separates the isobaric ions pamaquin, oxycodone and clonazepam
Representative chromatograms for pamaquinwith and without interferencesLC–MS/MS LC–FAIMS–MS/MS
1500
1000
500
0543210
800
400
0543210
1500
1000
500
0543210
800
400
0543210
10pg pamaquin
10pg pamaquin50ng oxycodone2.5ng clonazepam
Inte
nsity
(cps
)
Time (min)
10pg pamaquin
10pg pamaquin50ng oxycodone2.5ng clonazepam
interference no interference
• Using FAIMS, no interference of oxycodone or clonazepam on pamaquin• Using FAIMS, signal for pamaquin increased
Problem: Separation of positional isomers
theophylline paraxanthine
LC-ESI-MS analysis – ions co-elute ESI-FAIMS-MS – ions separated
6050403020100
43210
ParaxanthineTheophylline
Inte
nsit
y (c
ps x
10
-3)
Time (min)
100
80
60
40
20
0201510
Theophylline
Paraxanthine
Compensation Voltage
Inte
nsit
y (c
ps x
10-3
)
Problem: Separation of diastereomers
-8-6-4-20
Compensation Voltage
NPE
NE
PE
E
MEMPE
CH3
OH
NHCH3
CH3
OH
NH2
CH3
OH
NCH3 CH3
CH3
OH
NHCH3
CH3
OH
NH2
CH3
OH
NCH3 CH3
(-)-ephedrine (E)
(+)-pseudoephedrine (PE)
(-)-methylephedrine (ME)
(+)-methylpseudoephedrine (MPE)
(-)-norephedrine (NE)
(+)-norpseudoephedrine (NPE)
McCooeye, M.A.; Ding, L.; Gardner, G.J.; Fraser, C.A.; Lam, J; Sturgeon, R.E.; and Mester, Z.; Anal. Chem., 2003, 75, 2538-42.
Problem: Separation of diastereomers in presence of an endogenous interference
100
0
% In
tens
ity
-10-8-6-4-20Compensation Voltage
phospholipidinterference
b
a
m/z 497 → 163
• Diastereomers a and b are difficult to separate chromatographically, and the endogenous compound makes the background high• FAIMS allows the diastereomers to be analyzed in the presence of the endogenous interference
Problem: Removal of endogenous interferenceEstradiol-3-Sulfate (APCI)
• Many peaks present in LC-APCI-MS chromatogram - difficult to determine which is estradiol-3-sulfate• Major peak in LC-APCI-FAIMS-MS is estradiol-3-sulfate
Estradiol-3-Sulfate (TurboIonSpray)
interference
• LC-MS/MS of estradiol-3-sulfate in dilute urine shows endogenous interference present•FAIMS removes interference, quantitation is more accurate
4-Hydroxyisoleucine in rat plasma
3210 3210
LC-MS/MS LC-FAIMS-MS/MS30 pg on column
Problem: Removal of endogenous interference
• LC-MS/MS of 4-hydroxyisoleucine in plasma shows endogenous interference present; 0.1 amu resolution (i.e., H-SRM) still has interference •FAIMS removes interference, quantitation is more accurate•Chromatographic run time can be reduced
Problem: High backgroundLC-MS/MS vs. LC-FAIMS-MS/MS
50 pg/mL of drug + metabolite in plasma
LC-ESI-MS/MS S/N: 13
LC-ESI-FAIMS-MS/MS S/N: 30
LC-ESI-MS/MS S/N: N/AP
LC-ESI-FAIMS-MS/MS S/N: 30
Drug
Metabolite
• Drug shows a S/N improvement of ~2:1 with FAIMS• Without FAIMS, metabolite is not detected
Problem: Long analytical run time
Acetaminophen Cytochrome P450 1A2 Marker Assay
• Monitors the metabolism of phenacetin to acetaminophen
• Detect acetaminophen in the presence of a large excess of phenacetin
• Two challenges:– High background in liver microsome blank at the m/z for both
phenacetin and acetaminophen– Phenacetin undergoes CID at inlet to mass spectrometer to
form acetaminophen, affecting quantitation
FIA-SIM of human liver microsome blank after protein precipitation
APCI-MSm/z 180 (phenacetin)
m/z 152 (acetaminophen)
m/z 180 (phenacetin)
m/z 152 (acetaminophen) APCI-FAIMS-MS
• High chemical background by APCI-MS; chromatography used• FAIMS removes chemical background; chromatography not needed
Human liver microsomes after protein precipitation using FIA-SIM
5 µM Acetaminophen, 80 µM Phenacetin
phenacetin
acetaminophen
• FAIMS excludes phenacetin from the acetaminophen analysis• 5-minute LC separation is not required• Analysis time is reduced from 5 minutes to 1 minute
FAIMS – Robustness for batch analyses
FAIMS system run for 54 hours analyzing 1 ng/mL norverapamil(analyte) and verapamil (IS) in urine
Spiked urine diluted 1:3 with 0.1% formic acid in water (final conc. 250 pg/mL) 10 µL injected onto SB-Aq column
1.5
1.0
0.5
0.0
Are
a Ra
tio
(NV/
IS)
6005004003002001000Injection Number
•600 injections over a 54 hour period gave an RSD of 6.3%
FAIMS could save you months
Prepare Samples
Problem with LC-MS Method
Vary MS Conditions
Use FAIMS
Vary Chromatographic
Conditions
New LC-MS Method
analyze
solved
solved
solved
not solved
not solved
Save time, save money!
not solved
Assembly on an AB/Sciex API 3000
Control SoftwareIntegrated with Analyst 1.4.1
Waveform Generator
Electrode Interface Kit
Acknowledgements
Beata KolakowskiDavid BarnettGarnet McRaeJames KapronRandy Purves
Roger GuevremontUnny Thekkadath
Basis of FAIMS Operation• exploits differences in an ion’s mobility resulting from an
alternating high (Kh) and low (K) electric field
Increasing Electric Field Strength
1.05
1.00
0.95
A
B
C
Kh/K
Transport Properties of Ions in GasesE.A. Mason and E.W. McDanielJohn Wiley & Sons, Inc., 1988.
Recommended