Integrated SPE/LC/MS/MS Analysis of Small Molecules in Biological Samples

Kenneth C. Lewis OpAns, LLC

RTP, NC

Options for Analyte Extraction from Biological Fluids

Automating SPE

Example Options:On-Line 2D

• Cohesive (Turbo Flow), Waters (Oasis), Shimadzu (CoSense)

Off-Line• Automation of traditional tubes• Gilson, Hamilton, Tecan, Zinsser

On-Line Tube• Put a tube on an autosampler• HTA HT400E, Gerstel

On-Line Cartridge• Spark Holland (Symbiosis/Prospekt) • Microliter (ITSP)

Why ITSP

Options:Works on my current hardware (standard PAL)

• No capital investment• Familiar with software control

Wide variety of phases

Low volume device• 10 to 100 µL typical load• 40 to 100 µL typical elute

Flexible batch size

Reasonable consumable cost

In-Line process does not impact analysis time

How it works

Syringe Needle

PAL Needle GuideNeedle Centering Attachment

Septum

8mm Crimp Seal

PAL Needle Guide

Needle Guide

SPE or Filter Media

Funnel Cup

Typical ITSP Process

1. Aspirate conditioning solvent #12. Pick up device and move to Prep location3. Dispense solvent #1 4. Blow out with air5. Condition with solvent #26. Blow out with air7. Load Sample8. Wash with solvent9. Aspirate elution solvent10. Move device to elution vial11. Dispense solvent12. Blow out with air13. Discard the device14. Inject sample

Unique possibilities

Moving the deviceAllows multiple elutions

• Multiple elutions for multiple analytes• Multiple cleanup steps

Automated method development• In which fraction does the analyte elute?• Multiple solvent combinations.

Filtration• Pull sample through a filter• Multiple cleanup steps

Method Development

Samples

ITSP Devices

Elution Solvents

Fractions

Load Sample onto Device

Instrument

Versatility: In-Line and Off-Line

In-Line• Under MassHunter control

• Integrated Sample Prep and Analysis for automated analysis

• The efficient way to analyze samples.

Off-Line• Under CTC Cycle Composer control

• More flexible programming.

• Easier to optimize parameters (e.g. dispense rates)

• Since the QQQ is configured with the 1200 ALS, it can be running samples.

• The efficient way to develop methods.

Off-Line: Cycle Composer Control

Converting: Cycle Composer to MassHunter

Convert CycleComposer .pma file to MassHunter .cyx file

Converting: Changing Autosamplers

It is easy to change between the 1200 ALS and CTC PAL

In-Line: MassHunter Control

Immunosuppressants

Tacrolimus Cyclosporin A Sirolimus

Purpose:• SPE has been shown to be important for improved assay robustness 1

• Automation is needed to reduce analysis/turn-around times

• Evaluate: Recovery, Reproducibility, Ruggedness, Overall Performance

1Annesley TM. Clin Chem 2005;51:457-60

Immunosuppressants: ITSP Method

Sample: Start with whole blood extract 1

System: CTC Analytics HTC PAL with a 100 μSyringe and 3 cold stack traysITSP cartridges: SPE uLplate C8 (10 mg), Product No. 07-C810-20ASolvent A: Acetonitrile Solvent B: 50% Methanol in AcetonitrileSolvent C: 10% Methanol in Water Solvent D: 30% methanol in water

Step Solvent Volume FlowrateClean SYR A 100uL x 2 SYR Max uL/secCondition B 100 20Condition C 100 20Aspirate Air 25 200

Load Sample 100 5Aspirate Air 25 10

Clean SYR A 100uL x 2 SYR MaxWash D 100 10

Aspirate Air 25 Air FlushElute B 100 5

Aspirate Air 25 SYR Max

1Napoli KL. Ther Drug Monit 2006;28:726-36.

Immunosuppressants: Analytical Method

Solvent A: 2mM ammonium acetate and 0.1% formic acid in waterSolvent B: 2mM ammonium acetate and 0.1% formic acid in methanolColumn: Agilent Eclipse XDB-C8 Rapid Resolution (2.1 x 30 mm, 3.5μColumn Temp: 60ºC; Injection Vol: 15 uL; Flowrate: 0.5 mL/minGradient: Time (min) %B

0.0 501.0 100 (step)2.0 50 (step)3.0 50

Ionization: ESI (positive ion); Detection: Multiple Reaction MonitoringTransitions: Ascomycin (IS): m/z 809.4>756.5

Tacrolimus: m/z 821.4>768.5Desmethoxy-rapamycin (IS): m/z 901.4>834.6Sirolimus: m/z 931.4>864.6Cyclosporine: m/z 1219.7>1203.0Cyclosporin G (IS): m/z 1233.7>1217.0

LC: Agilent Model 1200 SLMS: Agilent Model 6410 HotBox QQQ

Immunosuppressants: Cyclosporine A

Cyclosporine (10 ng/mL)

Cyclosporine (1500 ng/mL)

CSG (CSA IS)

CYCLOSPORINE CONCENTRATION (ng/mL)

16001400120010008006004002000

AR

EA

RA

TIO

(CS

A to

CS

G)

4.0

3.0

2.0

1.0

0.0

CYCLOSPORINE

Y=0.00228X + 0.012

r**2= 1.000

CyclosporineLevel Within Total

1 (Low) 4.5 5.32 (Mid) 3.2 3.83 (High) 2.7 3.2

Immunosuppressants: Sirolimus

SirolimusLevel Within Total

1 (Low) 9.6 9.72 (Mid) 4.8 7.73 (High) 7.4 7.4

SIROLIMUS CONCENTRATION (ng/mL)

6050403020100

AR

EA

RA

TIO

(SR

L to

DM

RP

)

1.2

1.0

.8

.6

.4

.2

0.0

SIROLIMUS

Y=0.0234X - 0.002

r**2=0.996

Sirolimus (1.0 ng/mL)

DMRP (SRL IS)

Sirolimus (50 ng/mL)

Immunosuppressants: Tacrolimus

TacrolimusLevel Within Total

1 (Low) 1.8 2.32 (Mid) 1.3 3.43 (High) 1.9 2.7

TACROLIMUS CONCENTRATION (ng/mL)

6050403020100

AR

EA

RA

TIO

(TA

C to

AS

CO

)

1.5

1.2

.9

.6

.3

0.0

TACROLIMUS

Y=0.0263X + 0.000

r**2=0.999

Tacrolimus (1.0 ng/mL)

ASCO (TAC IS)

Tacrolimus (50 ng/mL)

Steroids

Purpose:• ITSP device seems to be a good fit for plasma volumes

• Use Steroids as a test case for direct injection of plasma

• Evaluate: Recovery, Reproducibility, Ruggedness, Overall Performance

CH3

CH3

OHOH

O

O

OHCH3

CH3

OHOH

O

O

O

CortisolC21H30O5

MW=362.5 Da

CortisoneC21H28O5

MW=360.5 DaUsed as Internal Standard

Steroids: Analytical Conditions

LC conditions – Agilent 1200SLSolvent A: Water with 0.05% Trifluoroacetic AcidSolvent B: Methanol with 0.05% Trifluoroacetic AcidColumn: Zorbax XDB C18 1.7 mm 3.0x30mm columnColumn Temp.: 55o CInjection Vol.: 5 μLFlowrate: 1.5 mL/minGradient: Time (min) %B

0.00 300.10 500.80 530.81 1001.00 1001.05 30

MS conditions – Agilent 1956B

Ionization: Multi-mode Source with APCI and ESIPolarity: PositiveDetection: Single Ion Monitoring

Cortisone (m/z 361), Cortisol (m/z 363)

Steroids: ITSP Conditions

1. The ITSP SPE cartridge was washed with 100 μL of Acetonitrile at 10μL/sec flow.

2. The ITSP was washed with 100 μL of Water (0.2% Formic Acid) at 10μL/sec flow.

3. 40 μL of sample (plasma) was loaded onto the ITSP at 5 μL/sec flow.

4. The ITSP cartridge was moved over collection vial 1 and washed with 30 μL of 20% Acetonitrile in water (0.2% FA) at 5μL/sec.

5. The ITSP cartridge was moved over collection vial 2 and eluted with 80 μL of 80% Acetonitrile in water (0.2% FA) at 5μL/sec.

6. The ITSP cartridge was moved over collection vial 3 and eluted with 80 μL of 100% Acetonitrile (0.2% FA) at 5μL/sec.

7. The vials were capped.

8. The vials were mixed.

9. The samples were analyzed using the LC-MS method.

10. Peak areas were determined using Chemstation software.

11. The following results were obtained for the second elution fraction (vial 2)

Steroids: Chromatograms

Steroids: Recovery and Linearity

Steroids: Precision

Steroids: Direct Injection of Plasma

Conclusions:• ITSP is a good fit for SPE of plasma

• Volumes match• Good recovery• Reproducible and Rugged

• Easy to Automate

Issues• Analyte stability in plasma during the sequence• Pipetting plasma on the PAL• When is the IS added?

Conclusions

We have successfully automated SPE LC-MS/MS using ITSP

Full prep + inject cycle takes 3 to 5 minutes.

Scaling from 1mL tubes to ITSP cartridges requires method refinement.

For plasma, stability and pipetting must be considered.

Urine works very well.

Acknowledgements

Agilent•Tony Brand•M. P. George

Microliter•Kim Gamble•Rick Youngblood

OpAns•Kimberly Eaton•Thurman Allsup•Laura Edwards•Kent Sowers

University of Wisconsin Hospitals•Gary Lensmeyer

Vitamin D

25-Hydroxyvitamin D2 25-Hydroxyvitamin D3

Vitamin D: ITSP Method

System: CTC Analytics HTC PAL with a 100 μSyringe and 3 cold stack traysITSP cartridges: ITSP Evolute C8 (10 mg), Microliter Product No. 07-BC10-20ASolvent A: Acetonitrile Solvent B: MethanolSolvent C: Water with 0.1% formic Solvent D: 50% methanol in water

Step Solvent Volume FlowrateClean SYR A 100 x 2 uL SYR Max uL/secCondition B 100 10Condition C 100 10Aspirate Air 50 15

Load Sample 100 5Aspirate Air 50 15

Clean SYR A 100 x 4 SYR MaxWash 1 C 100 10Wash 2 D 100 10Aspirate Air 50 15

Elute B 50 x 2 5Aspirate Air 50 15

Vitamin D: Analytical Method

Solvent A: 0.1% formic acid in waterSolvent B: 0.1% formic acid in methanolColumn: Agilent Zorbax Bonus RP (2.1 x 50 mm, 3.5μ)Column Temp: 30ºC; Injection Vol: 5 uL; Flowrate: 0.4 mL/minGradient: Time (min) %B

0.0-0.50 500.50-1.50 50-100 (linear)1.50-3.25 1003.25 503.35-5.00 50

Ionization: APCI (positive ion); Detection: Multiple Reaction MonitoringTransitions: 25-OH Vitamin D3: 401.3>159.3

25-OH Vitamin D2: 413.2>159.3Vitamin D3 (preliminary IS): 385.3>159.3

LC: Agilent Model 1200 Rapid ResolutionTM

MS: Agilent Model 6420 QQQ with Multimode source

Vitamin D: 25-OH D3

Concentration 25-OH Vitamin D3 (ng/mL)

100806040200

Area

Rat

io (2

5-O

H D

3 to

D3)

.7

.6

.5

.4

.3

.2

.1

0.0

25-OH Vitamin D3Y = 0.0076X + 0.0102r**2 = 0.997

Vitamin D: 25-OH D2

Concentration 25-OH Vitamin D2 (ng/mL)

100806040200

Are

a R

atio

(25

-OH

D2

to D

3)

.7

.6

.5

.4

.3

.2

.1

0.0

25-OH Vitamin D2Y = 0.0070X - 0.0131r**2 = 0.999