FAIMS: The First Step in Solving LC-MS Method Development

Challenges

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

Asymmetric waveform for FAIMS

-1500v

+4000v

0v

thigh

tlow

1.5 µsec

4 µsec

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

• Reduces background

• Increases throughput

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

Increases Selectivity

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

FAIMS - How does it help you?

Increases selectivity

Reduces background

• Increases throughput

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

FAIMS - How does it help you?

Increases selectivity

Reduces background

Increases throughput

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

FAIMS on the API 3000

FAIMSconfiguration

conventionalconfiguration

FAIMSelectrodes

Selectra on the API 3000

Selectra on the API 3000

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.

Assembly on a Waters Micromass Quattro micro

Control Software

Electrode Interface Kit

Waveform Generator