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Integrated SPE/LC/MS/MS Analysis of Small Molecules in Biological Samples
Kenneth C. Lewis OpAns, LLC
RTP, NC
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
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.
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: 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: 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