Rapid Quantitative Analysis of Analysis by LC-MS/MS for Clinical Research Analysis of Immunosuppressant Drugs in Blood and for Clinical Research Linda Côté Plasma Senior Application Specialist Agilent Technologies 1 For Research Use Only. Not for use in diagnostic procedures.
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Rapid Quantitative Analysis of
Analysis by LC-MS/MSfor Clinical ResearchAnalysis of
Immunosuppressant Drugs in Blood and
for Clinical Research
Linda CôtégPlasma Senior Application Specialist
Agilent Technologies
1
For Research Use Only. Notfor use in diagnostic procedures.
Objectives
During Todays webinar, we will be discussing: • A four minute research method for quantifying five immunosuppressive
drugs Cyclosporin A, everolimus, mycophenolic acid, sirolimus and tacrolimus
• A two minute research method for quantifying four immunosuppressive drugs CyclosporinCyclosporin AA, everolimuseverolimus, sirolimussirolimus andand tacrolimustacrolimus
• The use of back-flushing, matrix-stripping liquid chromatography to reduce the throughput of matrix to the mass spectrometer
• The use of the same hardware and reagents for both research methods allowing for the greatest flexibility while eliminating the need to maintain multiple configurations and solvents
For Research Use Only. Notfor use in diagnostic procedures.
Configuration for on-line sample cleanupMatrix stripping liquid chromatography which allows for l th h t t th t tcleaner throughput to the mass spectrometer
Position 1
Samples are injected onto a trapping column where the immunosuppressants areimmunosuppressants are retained and washed, reducing throughput of matrix to the mass spectrometer. p
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For Research Use Only. Notfor use in diagnostic procedures.
Configuration for on-line sample cleanup
Position 1 Position 2
Aft 0 5 i t l i it h d d l t l t d t l ti l l h f th
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After 0.5 minutes, a valve is switched and analytes are eluted onto an analytical column where further chromatography is performed.
For Research Use Only. Notfor use in diagnostic procedures.
Columns Temp: 60 °C Injection volume: 40 µL (2 µL for MPA)Needle Wash: 1:1:1:1 MeOH:ACN:IPA:H2O + 0.1% formic acid
10 seconds (60 seconds for MPA)Injector Temp: 4 °C S it hi V l 0 0 i t P iti 1Switching Valve: 0.0 minutes – Position 1
0.5 minutes – Position 22.4 minutes – Position 1
Mobile Phase: A: 10 mM NH4 Acetate + 0.2% Formic Acid in WaterB: 10 mM NH4 Acetate + 0 2% Formic Acid in MethanolB: 10 mM NH4 Acetate + 0.2% Formic Acid in Methanol
Pump Gradients: (followed by 30 seconds post time)
Loading Pump (1260): Analytical Pump (1290):
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For Research Use Only. Notfor use in diagnostic procedures.
Poroshell 120 Columns for HPLC and UHPLC:
Poroshell 120 is a high efficiency, high resolution column choice for enhancing productivity in LC and LC/MS
• Poroshell 120 Columns have:• 80-90% efficiency of sub 2um• At ~40-50% lower pressure• 2X efficiency of 3.5um (totally porous)• A 2um frit to reduce clogging
0.5um
u t to educe c ogg g• A 600 bar pressure limit for HPLC or
UHPLC use 1.7um
• The superficially porous particle is 2.7um witha solid core (1.7um) and porous outer layerwith a 0 5um diffusion path
0.5um
with a 0.5um diffusion path
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For Research Use Only. Notfor use in diagnostic procedures.
Poroshell 120 Resists Plugging with 2 um Frit Use with Plasma or Similar MatricesUse with Plasma or Similar Matrices
Column: Poroshell 120 EC-C18, 3.0 x 50mm, 2.7um LC: Agilent 1200 RRLC (SL) Sample: Precipitated Plasma: 2 parts Plasma: 7 Parts 20/80 Water-MeCN w/0.1 % Formic Acid with 1 Part Diflusinalin 50/50 Water-MeCN 10 ug/ml (Final concentration Diflusinal 1 ug/ml) Shaken and allowed to settle 10 minutesNot Centrifuged/ Not Filtered
Diflusinal in Plasma
380
400 200000
Not Centrifuged/ Not FilteredInjection Volume: 1ul injections
Solvent A: Water w/0.1 % TFASolvent B: MeCN w/0.08 % TFAFlow Rate 1 ml/min 1 ul injectionTime % B
260
280
300
320
340
360
140000
160000
180000Time % B0 200.5 900.6 901.1 202.5 20
140
160
180
200
220
240
Pres
sure
80000
100000
120000
Effic
ienc
y (N
)
End PressPlates
0
20
40
60
80
100
120
0
20000
40000
60000 Good lifetime with plasma samples!
01 501 1001 1501 2001 2501
Injections
0
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For Research Use Only. Notfor use in diagnostic procedures.
Ion source conditionsDrying gas temperature: 225°Cy g gas e pe a u e 5 CDrying gas flow: 9 L/minNebulizer pressure: 35 psiSheath gas temperature: 325°CSheath gas flow: 12 L/minSheath gas flow: 12 L/minCapillary voltage: 4000 VNozzle voltage: 300 VQ1/Q3 resolution: 0.7 unit ∆ EMV 200 V (0V for MPA)
Note: During research method development, MPA was evaluated in positive and negativenegative modemode andand foundfound toto performperform betterbetter iinn positivepositive modemode
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For Research Use Only. Notfor use in diagnostic procedures.
Internal Standards
Analyte Internal StandardCyclosporin A Cyclosporin DEverolimus AscomycinMycophenolic Acid Mycophenolic Acid-d3
For Research Use Only. Notfor use in diagnostic procedures.
MRM Transitions tablePrecursor ions are ammonium adducts (except MPA and MPA-d3)
C d P I P d I D ll F (V) CE(V)Compound Prec Ion Prod Ion Dwell Frag (V) CE (V)Cyclosporine D 1233.9 1216.9 10 175 12Cyclosporine A 1219.9 1202.8 10 170 12Everolimus 975.6 908.5 10 185 12Sirolimus 931.6 864.5 10 170 12Tacrolimus 821.5 768.4 10 170 16Ascomycin 809.5 756.4 10 175 16Mycophenolic Acid Gluc 514.2 207.0 10 95 36Mycophenolic Acid D 3242 2101 10 80 16Mycophenolic Acid D3 324.2 210.1 10 80 16Mycophenolic Acid 321.1 207.0 10 80 16
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For Research Use Only. Notfor use in diagnostic procedures.
Results
Cyclosporin D Everolimus Mycophenolic Acid Gluc
TacrolimusCyclosporin A Mycophenolic Acid-D3
ts
Sirolimus Mycophenolic AcidAscomycin
Cou
nt
(MPA-G*)
Retention Time (min)Retention Time (min)
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Due to the presence of mycophenolic acid glucuronide (MPA-G) in plasma, chromatography is particularly important to the research analysis of mycophenolic acid. MPA-G is susceptible to in-source fragmentation where glucuronide is easily lost. If these analytes are not separated by retention time, deglucuronidated MPA-G (MP(MPAA-G*)G*) cancan falselyfalsely elevateelevate thethe determinationdetermination ofof mycophenolicmycophenolic acidacid concentrationsconcentrations.
For Research Use Only. Notfor use in diagnostic procedures.
ResultsCalibration curves for five immunosuppressive drugspp g
(a) Cyclosporine A - 11 Levels, 11 Levels Used, 33 Points, 33 Points Used
0.8
0.9
1 y = 4.697286E-004 * x + 5.341801E-004R^2 = 0.99909117
- Calibrators prepared in whole blood or plasma are needed tobuild a calibration curve covering the linear range ofquantitationquantitation
- Internal standards are necessary for MS/MS quantitation
Controls prepared in whole blood or plasma are needed to- Controls prepared in whole blood or plasma are needed toverify suitability of the analytical method
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For Research Use Only. Notfor use in diagnostic procedures.
- Calibrators prepared in whole blood or plasma are needed tobuild a calibration curve covering the linear range ofquantitationquantitation
- Internal standards are necessary for MS/MS quantitation
Controls prepared in whole blood or plasma are needed to- Controls prepared in whole blood or plasma are needed toverify suitability of the analytical method
However there are multiple choices available andHowever, there are multiple choices available and a decision on which one to use has to be made based on price and on regional availabilityp g y
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For Research Use Only. Notfor use in diagnostic procedures.
For Research Use Only. Notfor use in diagnostic procedures.
Calibrators, Internal Standards, ControlsChoices for this projectp j
ResearchMethod
Calibrators Internal Standards Controls
Four minute method
- Home made in whole blood withCerilliant (cyclosporin A,
- Cerilliant for MPA-d3- Sigma for ascomycin
NAmethod ( y p ,
sirolimus and tacrolimus) andSigma (everolimus) referencestandards
- Home made in plasma with
g y- Santa Cruz
Biotechnologies forcyclosporine D
pCerilliant (MPA and MPA-Gluc)reference standards
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For Research Use Only. Notfor use in diagnostic procedures.
Calibrators, Internal Standards, ControlsChoices for this projectp j
Method Calibrators Internal Standards Controls
Four minute method
- Home made in whole blood withCerilliant (cyclosporin A,
- Cerilliant for MPA-d3- Sigma for ascomycin
NAmethod ( y p ,
sirolimus and tacrolimus) andSigma (everolimus) referencestandards
- Home made in plasma with
g y- Santa Cruz
Biotechnologies forcyclosporine D
pCerilliant (MPA and MPA-Gluc)reference standards
Two minute th d
- Home made in whole blood withCerilliant (cyclosporin A
- Sigma for ascomycinSanta Cruz
Collaboratormethod Cerilliant (cyclosporin A,
sirolimus and tacrolimus) andSigma (everolimus) referencestandards
- Santa Cruz Biotechnologies for cyclosporine D
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For Research Use Only. Notfor use in diagnostic procedures.
Collaborator results for controls for the TwTwoo minuteminute research methodmethod
Compound Target (ng/mL) Mean (ng/mL)n=45
% of Target CV (%)n=45
Cyclosporine A95.6 95.6 100.0 6.3
187.0 197.6 105.6 4.9307.0 321.6 104.8 4.8
Sirolimus5.1 4.8 93.6 13.98.5 8.6 100.9 11.5
17.3 17.9 103.4 10.44 2 4 5 108 1 7 4
Tacrolimus4.2 4.5 108.1 7.47.6 7.7 101.3 6.6
12.5 13.1 104.9 7.9
Notes:Notes:Data acquired over 14 days by 4 different operators (45 measurements)Reference calibrators: ChromSystemsControls: BioradInternal Standards: ascomycin (Enzo Life Sciences), sirolimus-d3 (Medical isotopes),
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cyclosporine A-d4 (Toronto Research Chemicals)
For Research Use Only. Notfor use in diagnostic procedures.
Tips and tricks
• Sodium adducts vs ammonium adducts
• Washes at the end of a batch
• Vial size and pre-slit septa
• Mobile phase quality (grade and freshness)
• Inline filter between needle seat and injection valve
• Pipetting technique: manual vs electronic pipettesPipetting technique: manual vs electronic pipettes
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For Research Use Only. Notfor use in diagnostic procedures.
Supplies
SSupplier: Part number: Description:Agilent 821125-936 Trapping Column: Zorbax Eclipse Plus C18,
2.1x12.5mm, 5 µMAgilent 820888-901 Guard column hardware kit for trapping
For Research Use Only. Notfor use in diagnostic procedures.
Review and Summary
• Successful four research minute method for quantifying five immunosuppressive drugs Cyclosporin A, everolimus, mycophenolic acid, sirolimus and tacrolimus
• High throughput two research minute method for quantifying four immunosuppressiveimmunosuppressive drugsdrugs CyclosporinCyclosporin AA, everolimuseverolimus, sirolimussirolimus andand tacrolimus
• Back-flushing, matrix-stripping liquid chromatography to reduce the throughput of matrix to the mass spectrometer
• UU ising ththe same hharddware andd reagentts ffor bbothth research meththodds allllowiing ffor ththe greatest flexibility while eliminating the need to maintain multiple configurations and solvents
• Typypical pperformance results for both methods are well within accepptance criteria
• Choices for Calibrators, Internal Standards and Controls
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For Research Use Only. Notfor use in diagnostic procedures.
Acknowledgements
• Kevin McCann• LC/MS Application Champion, Agilent Technologies, Santa Clara, California, USA
• Andre Szczesniewski• LC/MS Application Specialist, Agilent Technologies, Schaumburg, Illinois, USA
• Peter Christensen• LC/MS Application Specialist, Agilent Technologies, Cheadle, UK
• DennisDennis--AlbertAlbert NagtalonNagtalon• Program marketing manager, Agilent Technologies, Santa Clara, California, USA
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For Research Use Only. Notfor use in diagnostic procedures.