using mass spectrometry in diagnosis &...
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Using Mass Spectrometry in Diagnosis &
Management
James C. Ritchie, Ph.D, DABCC, FACB
Director
Core & Special Chemistry LaboratoriesCore & Special Chemistry Laboratories
2011 ASCP Annual Meeting1700 – 1800
October 20th, 2011Veronese 2503
Disclosures
James C Ritchie, Ph.D.
• Research / Educational Grants
– BeckmanCoulter, Inc
– Abaxis, Inc
– Abbott Diagnostics
– Roche Diagnostics
– Waters, Inc.
• Federal Grants:
– R01-71531- Collaborative R01, BPD in Pregnancy
– P50-77083 - Predictors of Antidepressant Response (CIDAR)
– P50-77928 - Perinatal Stress: Pathways to Vulnerability (TRCBS)
– MH-69056 - Emory / GSK/ NIMH Collaborative Mood Disorders Initiative
– MH-078105 - Early Experience, Stress and Neurobehavioral Development
Center
Discussion Today
• MS Technology Primer
• Problems unique to LC-MS/MS
• One Lab’s Initial Experience
• New tools for old problems (new assays, etc)• New tools for old problems (new assays, etc)
• What’s Coming
• Summary
Fear Factors in the Clinical Lab
• 1970’s – Immunoassay
• 1980’s – PCR
• 1990’s – PGx • 1990’s – PGx
• 2000’s -Mass Spectrometry
Introduction to Mass Spectrometry
CH3COCH3CH3COCH3 CH3+COCH3CH3+COCH3 CH3C
+OCH3CH3C+OCH3
+CH3+CH3
+COH+COH
Mass Spectroscopy
Sample
Inlet
Sample
Inlet
Ionization
& Adsorption
of Excess Energy
Ionization
& Adsorption
of Excess Energy
Mass AnalysisMass Analysis
+COCH3+COCH3
Fragmentation
(Dissociation)
Fragmentation
(Dissociation)
DetectionDetection
Electron Ionisation Electron Ionisation
Electron
Trap
Ion Repeller Mass AnalyserIon Repeller
Sample
MoleculesElectrons
Ions
Chemical Ionization
• Reagent gas enters before samples (methane)
• An electron beam ionizes the reagent gas creating gas ions
• When sample molecules enter the source they collide with the reagent gas resulting in a collide with the reagent gas resulting in a charge transfer from the gas ions to the sample molecules.
• This is a valuable as it creates a “soft, non-fragmenting” ionization.
+
+
+
+
+ +
Matrix Assisted Laser Dissorption Ionization (MALDI)Matrix Assisted Laser Dissorption Ionization (MALDI)
Laser
e.g. N2 (354nM)
Nd-YAG (266nM), etcDesorbed
sample and
matrix ions
UV-adsorbing matrix
mixed with sample
Surface Enhanced Laser Desorption Ionization (SELDI) = a form of MALDI employing selective surfaces to pre-select compounds of interest
Mass spectrometry
CH3COCH3CH3COCH3 CH3+COCH3CH3+COCH3 CH3C
+OCH3CH3C+OCH3
+CH3+CH3
+COH+COH
Sample
Inlet
Sample
Inlet
Ionization
& Adsorption
of Excess Energy
Ionization
& Adsorption
of Excess Energy
Mass AnalysisMass Analysis
+COCH3+COCH3
Fragmentation
(Dissociation)
Fragmentation
(Dissociation)
DetectionDetection
Mass Analyzers
Two Classes:
• Beam-type
– Ions make one trip through the instrument and then strike the detector where they are destructively strike the detector where they are destructively detected (micro to milliseconds).
• Trapping–type
– Ions are held in a confined space by a combination of magnetic, electrostatic, and /or RF electrical fields. Fields are then manipulated to allow ions to be released (seconds to minutes)
Quadrupole Theory
In a quadrupole instrument, only electric fields are used to separate
ions according to mass, as they pass along the central axis of four
parallel, equidistant rods (poles) which have fixed (DC) and
alternating (RF) voltages applied to them.
Quadrupole TheoryPre-filter Quadrupole Mass Filter Stable Trajectory
Unstable Trajectories
•Only ions with the correct m/z values have stable trajectories within
an RF/DC quadrupole field.
• Ions with unstable trajectories collide with the rods, or the walls of the
vacuum chamber, and are neutralised.
•These stabilities are governed by the Mathieu equations.
Principles of Time-of-Flight
BAtm +=
m
eVv
acc2
=
2
2
1mveV
acc= mass
time
Source Detector
Time-of-Flight Mass Spectrometry
Basic TOF Instrument
Q-TOF Illustration
Ion Trap Mass AnalyzerIon Trap Mass Analyzer
• Ion traps are ion trapping devices that make use of a three-dimensional quadrupole field to trap and mass-analyze ionsmass-analyze ions
• invented by Wolfgang Paul (Nobel Prize1989)
• Offer good mass resolving power
FTFT--ICRICRFourierFourier--transform ion cyclotron resonancetransform ion cyclotron resonance
• Uses powerful magnet (5-10 Tesla) to create a miniature cyclotron
• Originally developed in Canada (UBC) by A.G. Marshal in 1974Marshal in 1974
• FT approach allows many ion masses to be determined simultaneously (efficient)
• Has higher mass resolution than any other MS analyzer available
LTQ Orbitrap Mass Spectrometer
Olsen, JV, et al, Molecular & Cellular Prot, 2009,8; 2759
IonizationIonizationMass
analysis 2Mass
analysis 2FragmentationFragmentation
Massanalysis 1Mass
analysis 1
MSMSMSMS
MS/MS vs GC-MS
InjectionInjectionMass
analysisMass
analysisIonizationIonizationGC
SeparationGC
Separation
INLETINLET SEPARATESEPARATE IDENTIFYIDENTIFY
GCGC MSMS
INLETINLET SEPARATESEPARATE IDENTIFYIDENTIFY
Tandem QuadrupoleInstrument Design
MS1 MS2Collision
Cell
Argon gasArgon gas
Precursor(s)Precursor(s)Product(s)Product(s)
Multiple Reaction MonitoringMRM
Both the first and second quadrupole mass analyzers are
held Static at the mass-to-charge ratios (m/z) of the
precursor ion and the most intense product ion,
respectively.
Both the first and second quadrupole mass analyzers are
held Static at the mass-to-charge ratios (m/z) of the
precursor ion and the most intense product ion,
respectively.
Static (m/z 315.1) Static (m/z 109.0)
Precursor(s)Precursor(s)
Capillary
~3kV
Capillary
~3kV
Ions evaporate
from the
Ions evaporate
from the
Electrospray Ionisation Electrospray Ionisation
~3kV~3kV
As droplet evaporates, the
electric field increases and ions
move towards the surface.
As droplet evaporates, the
electric field increases and ions
move towards the surface.
from the
surface
from the
surface
Electrospray or Atmospheric Pressure Chemical Ionization (APCI)
Issues Unique to LC-MS/MS Analyses
• Matrix Effects
• Differential ionization of Internal Standards or Calibrators
• In Source Transformation (fragmentation)
• Isobaric Compounds and Isomers• Isobaric Compounds and Isomers
• Cross-Talk effects
• Chromatographic Resolution
• Carry-Over
Vogeser & Seger, 2010, Clin Chemhttp://www.clinchem.org/cgi/doi/10.1373/clinchem.2009.138602
Sample Preparation
• Matrix: Serum, whole blood, urine
• Pretreatment process
– Protein Precipitation Protocols: Fast & Easy. However
associated with longer periods of ion suppression due associated with longer periods of ion suppression due
to early eluting, low molecular weight matrix
constituents
– Solid-Phase Extraction & Liquid-Liquid Extraction:
Slower & more chance for error. Do have shorter
periods of ion suppression.
Vogeser & Seger, 2010, Clin Chemhttp://www.clinchem.org/cgi/doi/10.1373/clinchem.2009.138602
In Source Transformation:
• Fragmentation occurring after column separation but before collision cell– MAP-G > MPA > MPA Fragment
– MPA-BE > MPA > MPA Fragment
• Can be a problem with endogenous drug metabolites– Check real patient samples in extended
chromatographic runs
Isobaric Compounds & Isomers
• Extremely important when measuring
endogenous analytes
• Often mandates complete chromatographic
resolution
Cross-Talk•Occurs when several mass transitionswith identical product ions are acquiredwith identical product ions are acquiredover a short time interval.
•If collision cell does not empty completely spurious signals can be recorded in a subsequent trace
•Common when several metabolites of a single drug are detected with identical fragment ions
•Solution: Increase interscan delay
“Analyte co-elution is generally
feasible due to the high
selectivity of SRM/MRM
experiments, if co-elution of
isobaric analyte isomers or ion
source fragmentation of the
analyte metabolites can be
ruled out”ruled out”
Vogeser & Seger, 2008, Clin Biochem, 41:649-662
Carryover
The AOI at 500 ng/mL causes a 331% Carry-Over.
10 ng/mL 100 ng/mL 500 ng/mL
Plasma 1 537 451 517
Plasma 2 539 481 410
Analyte 108000 1010000 4700000
Plasma 3 529 737 1660
Plasma 4 495 460 481
%Carryover = (Aavg – Bavg)/Bavg x 100
A = plasma pool 3, 5 ,7 B = plasma pool 2, 4, 6, 8.
Plasma 4 495 460 481
Analyte 107000 978000 4660000
Plasma 5 464 831 1980
Plasma 6 522 581 528
Analyte 105000 985000 4550000
Plasma 7 445 916 2900
Plasma 8 425 539 575
Analytical Sensitivity
• Limit of Absence (LOA)
– 20 replicates of zero calibrator or specimens without analyte run across multiple days
• LOA = mean + 2SD or + 3 SD
• Limit of Detection
– Measure specimens with levels (naturally, diluted or spiked) that – Measure specimens with levels (naturally, diluted or spiked) that approximate the LOA, but are consistently detectable
• LOD = mean of detectable concentration + 2SD or +3 SD of specimens
• Also, noise x 3
• Limit of Quantification (Functional Sensitivity)
– Minimum concentration where concentration can be measured reliably
• Imprecision CV > 20%
• Noise x 10
• Goals: Must be below clinical needs
ThroughputSelectivity
Requirements of Clinical Laboratory Methodology
Automation
Ease of Use
Sensitivity
Accuracy
Emory Transplant Center
�Region’s largest and only comprehensive organ and tissue transplant program.
In 2010 the Emory Center performed a total of 453 transplants:transplants:
160 Kidney22 Heart80 Liver26 Pancreas24 Lung128 Stem Cell13 Islet Cell
Choosing if Mass Spectrometry is Right for Your Lab
� Define your assay requirements
� Define your analytes and understand any alternative approaches
� Identify a mass spectrometer
� Test compounds
� Visit other labs
Can You Measure Your Analyte of Interest Using
Mass Spectrometry?
• Do you have access to the AOI?
• Have others done this already?
20
30
40
50
# o
f P
ub
lica
tio
ns
• Does it “fly” (ionization mode)?
• Are the calibrators/IS available?
• Is it financially viable?
• What are the clinical needs?
0
10
2000 2001 2002 2003 2004 2005 2006 2007 2008 2009
Year
# o
f P
ub
lica
tio
ns
2003: What Were Other Labs Doing for IS Drugs?
Reference Laboratories Methodology
Quest HPLC and TDx (by request)
Mayo Labs HPLC + LC- MS/MS
Lab Corp LC- MS/MS
ARUP LC - MS/MS
Academic Centers Methodology
MUSC LC - MS/MS
UNC LC - MS/MS
Univ. of Mich. LC - MS/MS
Univ. Penn HPLC (LC - MS for C2 protocols)
Univ. Washington (Seattle) Children’s
TDX
Duke LC - MS/MS
2003
Immunosuppressant Testing
Measurand Volume / yr Cost / Analysis Cost / yr
Cyclosporine 7588 $10.80 $81,950
Tacrolimus 13120 $12.00 $157,440
Sirolimus 744 $ 57.45* $42,743
TOTALS 21,452 $282,133
• Rapamycin TAT 1 to 4 days
• Expecting volume to increase substantially in future
• Each drug is a separate analysis, no opportunity for multiplexing
• Total cost to system over 7 years = $1,974,931
TOTALS 21,452 $282,133
*Sendout-Mayo Labs
LC-MS/MS
Proposal
Measurand Volume / yr Cost* / Analysis Cost / yr
Cyclosporine 7588 $6.35 $48,184
Tacrolimus 13120 $6.35 $83,312
Rapamycin 744 $6.35 $4,724Rapamycin 744 $6.35 $4,724
TOTALS 21,452 $136,220
* Cost includes reagents, labor, & instr. depreciation
• All drug TATs within 1 day• Capable of expanding to meet increased need• Runs are multiplexed • Total cost to system over 7 years = $953,540• Saving to system over 7 years = $1,021,391• Provides opportunities to do other assays
Process for Developing a Clinical Method
• Method Selection
• Selection of Key Operator• Outline/plan methods• Acquisition of materials• Set quality goals
Make a Implementation Plan Validate the Method
• Imprecision• Recovery• Linearity• Sensitivity• Specificity• Set quality goals
• Validation• Monitoring and statistics• SOP preparation, staff training
• Specificity• Interferences• Specimen• Method Comparison• Assay Calibration
LC-MS/MS Analyses
• Most current methods are home-brews
– Resources:
• Develop from scratch
• Previously validated method from colleagues
• Literature
• Vendors
• Most variation between labs related to differences in methods &
standards
• CLIA – High Complexity
Guidance Documents
• EU Directive 2002/657/EC
• FDA Guidance 118
• FDA Special Controls Guidance for Particular Drugs
• CAP Chemistry & Toxicology Checklist
• CLSI Guidance EP10-A3E
• SOFT and AAFS Guidelines
• WADA Identification Criteria
• New York State – Clinical Laboratory Standards of Practice
Guidance Documents
• EU Directive 2002/657/EC
• FDA Guidance 118
• FDA Special Controls Guidance for Particular Drugs
• CAP Chemistry & Toxicology Checklist
• CLSI Guidance EP10-A3E
• SOFT and AAFS Guidelines
• WADA Identification Criteria
• New York State – Clinical Laboratory Standards of Practice
Useful ResourcesCLSI Guidelines
– EP10 Preliminary Evaluation of Quantitative Clinical Laboratory
measurement Procedures
– EP15 User Verification of Performance for Precision and Trueness
– EP05 Evaluation of Precision Performance of Quantitative – EP05 Evaluation of Precision Performance of Quantitative
Measurement Methods
– EP06 Evaluation of the Linearity of Quantitative Measurement
– EP09 Method Comparison and Bias Estimation
– GP31 Laboratory Instrument Implementation, Verification, and
Maintenance
– EP50 Mass Spectrometry in the Clinical Laboratory
EP50 Mass Spectrometry in the Clinical
Laboratory
• General overview of mass spectrometry and clinical applications
• Guidelines on analytical method development and validationand validation
• Seeks to harmonize some of the international documents
• http://www.clsi.org/source/orders/free/C50-A.pdf
Immunosuppressant Drugs by LC/MS/MS –
Protocol (circa 2004)
Microfuge tube protocol.
1. In a 1.5mL Eppendorf tube accurately pipette:50µL Lysis Solution A (0.4M ZnSO4)
200µL whole blood (Calibrators, QCs or patient samples)
2. Briefly vortex mix samples (5 – 10sec)
3. Add 500µL of Precipitating Solution (25ng/mL Ascomycin + 100 3. Add 500µL of Precipitating Solution (25ng/mL Ascomycin + 100 ng/mL CycloD in acetonitrile)
4. Vortex mix for approximately 1 minute or until the entire sample is thoroughly mixed
5. Centrifuge for 5 minutes at 14,500rpm
6. Cut top off of micro-centrifuge vial. Place vial into HPLC autosampler
7. Inject 10µL on the LC-MS/MS system. Use C-18 column (4X3mm –Phenemonex)
Immunosuppressant Drugs by LC/MS/MS -
Protocol
Compound
Precursor Ion
Daughter Ion
Cyclosporine A 1220 1203
Tacrolimus 821.5 768.5Tacrolimus 821.5 768.5
Rapamycin 931.6 864.5
Ascomycin 809.5 756.4
Cyclosporine D 1234.1 1217.2
Cyclosporine A & D
Immunosuppressant Standard (20/20/500 ng/mL)
Ascomycin, FK-506, Rapamycin
Immunosuppressant Standard (20/20/500 ng/mL)
Cyclosporine D
Cyclosporine A
Rapamycin
FK-506
Ascomycin
Immunosuppressant Drugs by LC/MS/MS -
Limits
Compound
LOD
(ng/mL)
LOQ
(ng/mL)
Linearity
(ng/mL)
Tacrolimus 0.1 0.6 40
Cyclosporine A 3.0 10 1000
Rapamycin 0.3 0.6 40
Immunosuppressant Drugs by LC/MS/MS -
Imprecision
Interassay
Tacrolimus Cyclosporine A Rapamycin
Concentration 4 90 4Concentration
(ng/mL)
4 90 4
%C.V. 7 10 13
Intra-assay
%C.V. 2 7 9
Matrix effects
1. Prepare extracts on 5 random blood samples from patients not receiving an IS drug.
2. Prepare 2 extracts using water as matrix.
3. To 500 uL of each extract add 150 ng Cyclo A, 75 ng Cylo D, 15 ng FK-506, 15 ng Rapa, and 20 ng Asco (all in 50ul).
4. Compare whole blood recoveries from water.
FK-506(Area)
Cyclo A(Area)
Rapa(Area)
Asco(Area)
Cyclo D(Area)
Water 3651 75885 1267 8578 36918
#1 4241 77293 1580 8650 38890
#2 4834 71217 1317 8787 34821
#3 4486 73005 1359 8218 27974
#4 4836 71500 1360 9023 36014
#5 4394 75963 1402 8225 32160
Blood mean 4559 73796 1404 8581 33972
Recovery 125% 97% 111% 100% 92%
AMR & CRR
AMR(ng/mL)
CRR(ng/mL)
Cyclosporine A 100 - 1000 10 - 2000Cyclosporine A 100 - 1000 10 - 2000
FK-506 0.3 - 40 0.3 - 80
Rapamycin 0.6 - 40 0.6 - 80
LC/MS/MS - Throughput
• Run time = 2 mins/sample
• Cycle time = 3.5 mins.
• One run contains 18 samples + 4 stds + 3 • One run contains 18 samples + 4 stds + 3 controls.
• A run takes 87.5 mins.
• 5 runs can be performed in 7.3 hours.
• This means a maximum of 90 specimens can be analyzed per shift per instrument.
Emory Medical Laboratories
Immunosuppressant Monitoring
• Average monthly workload for 2004:
» Tacrolimus – 1322
» Cyclosporine A – 732
» Sirolimus – 426» Sirolimus – 426
This equals 2480 samples a month or 83 samples per day
• Analysis performed in the Special Chemistry
Section, 7 days per week, dayshift only.
Billboards You’ll Never See:Billboards You’ll Never See:
RAPA – Assay Comparison
(both LC/MS/MS)
Demming Regression:Emory = 1.006 Mayo + 0.05R = 0.9903N = 24Average Bias = 0.10
FK-506 Assay Comparisons
Demming Regression:LC/MS/MS = 1.022 IMx-0.117R = 0.9380N = 39Average Bias = 0.064
400
500
600
HPLC (Mayo)
(ng/mL)
CyclosporineLC/MS/MS vs HPLC
n=19 random patient specimens
LCMS HPLC100 114200 215400 416
HPLC = 1.0056 LCMS + 13.548R2 = 0.9574
0
100
200
300
0 100 200 300 400 500 600
HPLC (Mayo)
(ng/mL)
LC/MS/MS (Emory)(ng/mL)
How does immunoassay compare to
LC-MS/MS?
Pure Compound:Pure Compound:
1. LC/MS/MS = 0.912Abbott -2.071, r = 0.9122. On average the LC/MS/MS value is 9.7% lower than the Immunoassay .3. Immunoassay Therapeutic Range was 50 to 400 ng/mL4. From this comparison, LC/MS/MS Therapeutic Range is 43 to 363 ng/mL
Patient Sample Comparison
Emory Cyclosporine Assay Comparison
(using patient samples)
1500
2000
2500
LC/MS/MS
n=185 patient samples w ith complete data as of 12/16/03
LC/MS/MS = 0.789TDx - 46.752
R2 = 0.9011
0
500
1000
0 500 1000 1500 2000 2500
Immunoassay (Abbott Monoclonal)
LC/MS/MS
n=185 patient samples with complete data as of 12/15/03 Therapeutic Range comparable to TDx assay = -7 to 269ng/mL
On average the LC/MS/MS values are 44% lower than the immunoassay values
• Cyclosporine (CsA) is a cyclic undecapetide of MW 1203.6 Da.
• It is used primarily in solid organ and bone marrow transplantation.
Cyclosporine
transplantation.
• CsA exerts its immunosuppressive effect by binding to cyclophilin, inhibiting calcineurin, and halting T-cell receptor transcription of the IL-2 gene.
• CsA is metabolized by Cytochrome P450 -3A4 and 3A9, in the liver, small intestine, and kidney .
Cyclosporine
• At least 30 metabolites (hydroxylated, demethylated, cyclized, and oxidative by-products) have been identified.
• Metabolites AM1 and AM9 have approx. 14 to 16% of the activity of the parent drug (in the MLC assay). Little is known about the activity of other metabolites.
• The above two metabolites are seen at 150 and 75% concentrations relative to the parent drug at steady state in kidney transplant patients.
Cyclosporine
• Therapeutic drug monitoring is mandated as bioavailability, metabolism, and excretion can differ markedly among individuals.
• Additionally, nephrotoxicity, hepatoxicity, and neurotoxicity can occur with overdosage whereas graft rejection can result from underdosage.
• The current recommendation for CsA monitoring is to analyze trough concentrations of the drug in EDTA whole blood using a method specific for the parent compound. Monitoring of the active metabolites is not currently recommended. (Oellerich et al, Consensus Conference
on CsA Monitoring in Organ Transplantation:report of the consensus panel. Ther. Drug Monit. 1995, 17:642-654.)
Metabolic Pathway of Cyclosporine A
AM1, AM9, AM4n are the primary metabolites found in human blood and urineOther metabolites result from further metabolism of the primary metabolites.
Metabolite Toxicity ?
• Toxic mechanisms of CsA and its metabolites are not
necessarily identical. No good models of organ specific
toxicity exist for CsA.
• CsA metabolites are in general less toxic than parent CsA in
rat models or in the renal epithelial cell line (LLC-PK ).rat models or in the renal epithelial cell line (LLC-PK1).
• Clinical reports have not shown a clear association between
blood concentrations of CsA metabolites and neuro- /
nephrotoxicity.
Update Memo
Moving CsA Testing to LC-MS/MS Assay
• “Methodology will change as of 11/24/03”
• “New Therapeutic Range:
43 to 363ng/mL (Specific for parent drug)”
• “Individual patient levels may vary by as much as 70%”
• “Recommend if see a drop of 30 to 40% and patient is stable, no change. If more than that proceed to increase dose slowly.”
Laboratory Update Memo:
Moving CsA Testing to LC-MS/MS Assay
Advantages:Advantages:– Measures 3 immunosuppressive drugs
(CsA, Tacrolimus, Sirolimus) simultaneously.
– Specific for only parent compound of each drug.– Specific for only parent compound of each drug.
– Corresponds very well to the HPLC method which has been
regarded as the “gold standard” method for CsA
determinations. (Holt, et al, Ther. Drug Monitor., 24:59-67, 2002)
– Increased lab efficiency, decreased costs, decreased reliance
on single vendor of immunoassay.
11/24/03
• Due to a distribution problem memo not
distributed until 11/26/03
Duplicate Patient Testing
• To help transition to the new methodology lab agreed to perform duplicate cyclosporine testing.
Transplant Type
# Patients Specimens
Heart 82 128
Kidney 231 247
Liver 23 41testing.
• 833 patient specimens were analyzed (12/15/03
thru 01/15/04).
Liver 23 41
Lung 23 243
Stem Cell & Bone Marrow
8 52
Pancreas 5 42
C2 Protocols
13 45
Total Data SetCyclosporine Patient Sample Comparisons
12/15/03 thru 01/15/04
1200
1400
1600
1800
2000
LC/MS Immuno
40 100
102 200
227 400
601 1000
LC/MS/MS = 0.6232TDx - 22.659
R2 = 0.9629
0
200
400
600
800
1000
0 500 1000 1500 2000 2500 3000
TDx - Immuno Assay
(ng/mL)
LC/MS/MS
(ng/mL)
Heart Transplant Specimens
Heart Transplant Specimens
n=128
350
400
450
500
LC/MS Immuno
44 100
97 200
203 400
522 1 000
LC/MS/MS = 0.5313TDx - 9.3919
R2 = 0.9157
0
50
100
150
200
250
300
0 100 200 300 400 500 600 700
TDx - Immuno Assay
(ng/mL)
LC/MS/MS
(ng/mL)
Kidney Transplant Specimens
Kidney Transplants
n=247
1000
1200
1400
LC/MS/MS (ng/mL)
LC/MS Immuno
41 100
104 200
231 400
613 1000
LC/MS/MS = 0.6362TDx - 23.124
R2 = 0.9669
0
200
400
600
800
0 200 400 600 800 1000 1200 1400 1600 1800 2000
TDx - Immuno Assay (ng/mL)
LC/MS/MS (ng/mL)
Individual Patient Data
0
200
400
600
800
1000
1200
1400
12/22/03
12/23/03
12/24/03
12/25/03
12/26/03
12/27/03
12/28/03
12/29/03
12/30/03
12/31/03
1/1/04
1/2/04
1/3/04
1/4/04
1/5/04
1/6/04
1/7/04
1/8/04
1/9/04
1/10/04
1/11/04
1/12/04
Cyclosporine (ng/mL)
TDx-Immunoassay
LC/MS/MS
Kidney Transplant Patient: PI (10/16/03)
12/22/03
12/23/03
12/24/03
12/25/03
12/26/03
12/27/03
12/28/03
12/29/03
12/30/03
12/31/03
1/10/04
1/11/04
1/12/04
Date
Individual Patient Data
0
50
100
150
200
250
300
350
12/26/03
12/27/03
12/28/03
12/29/03
12/30/03
12/31/03
1/1/04
1/2/04
1/3/04
1/4/04
1/5/04
1/6/04
1/7/04
1/8/04
1/9/04
1/10/04
1/11/04
1/12/04
Date
Cyclosporine (ng/ml)
TDx-Immunoassay
LC/MS/MS
Kidney Transplant Pat: AL (5/1/92)
New Transplant Specific
Ranges ?
Post Heart Transplant
Immunoassay
CsA
LC/MS/MS
CsA
(Based on sample
comparison)
LC/MS/MS
CsA
(Heart transplant group)
1 to 180 days 300 150 150
6 to 18 months 250 123 125
18 mos to 2 yrs 200 97 100
>2yrs 150 70 75
Conclusions
1. Developing an LC-MS/MS assay for clinical use is just like setting up any other assay in the lab.
2. Defining the process and selecting a good chief operator are key.
3. Today’s medical technologists are capable of understanding 3. Today’s medical technologists are capable of understanding and using LC-MS/MS for routine analyses
4. Standardizing the analytical process and system maintenance are crucial for success.
5. LC-MS/MS can be a cost effective tool in the clinical laboratory
Conclusions
6. Looking at the Emory transplant population LC-MS/MS
cyclosporine concentrations are 40 to 60% lower than the
corresponding immunoassay values (TDx)
7. Individual patients “track” consistently in the LC-MS/MS
assay
8. Analysis of cyclosporine A metabolites does not seem
warranted for the vast majority of patients.
9. It is not enough to just have better technology
… the best resources when switching assays are data and
good communication.
Can LC-MS/MS analysis of IS drugs be
considered a routine test?
1. Authors describe the analysis of tacrolims in their lab over a 4.5
yr period by four laboratory scientists.
2. During this period they ran 4029 batches which included 61,027
patient samples.
3. Accuracy was 97.6 to 98.5% and Imprecision was <8.0%3. Accuracy was 97.6 to 98.5% and Imprecision was <8.0%
4. Only four batch failures occurred out of the 4029 batches (0.1%
failure rate).
5. Of the 4 batch failures, 3 were due to errors attributed to the
scientist and 1 due to instrument malfunction.
These data document the ruggedness of the method and suggest it is
ideally suited as a routine test for tacrolimus in the clinical setting
P.J. Taylor, et al., J. Chromatogr. B (2011), doi:10.1016/j.jchromb.2011.06.024
Survey CSM-A 2011
CAP Proficiency Testing
• 480 labs participated:
Cyclo = 485, Tacro = 479, Rapa = 247
• 68 Labs used LC-MS/MS:• 68 Labs used LC-MS/MS:
Cyclo = 66, Tacro = 64, Rapa = 70
• Immunoassays:
Cyclo = 8, Tacro = 4, Rapa = 2
Results
Cyclosporine Mean
Immunoassay
% CV
LC-MS/MS
%CV
CS-01 199.65 ng/mL 9.2 9.1
CS-02 351.50 8.6 9.7
CS-03 78.93 12.9 9.0
TacrolimusTacrolimus
CS-01 8.49 ng/mL 13.4 8.6
CS-02 16.06 10.1 9.3
CS-03 4.00 22.3 9.6
Sirolimus
CS-01 10.82 ng/mL 7.4 15.9
CS-02 22.64 7.9 13.9
CS-03 4.28 11.6 16.3
K/DOQI – Classification of CKD based on GFR stratification
Renal Impairment
STAGE 1Kidney DamageNormal or
STAGE 2Kidney DamageMild Kidney
STAGE 3ModerateKidney
STAGE 4Severe Kidney
STAGE 5Kidney
Measuring Renal Function Using Iothalamate
Glomerular filtration rate (GFR) is the best estimate
of kidney function and determines your stage of
kidney disease (NKDEP).
Normal or Kidney Function
Mild Kidney Function
Kidney Function
Kidney Function
Kidney Failure
015306090120
Adapted from K/DOQIGFR
• Not bound by protein
• Freely filtered by glomerulus
• Ease of measuring
• Assay interferences
• Secreted by renal tubule
• 24 hr: creatinine creatine
• Possible incomplete and inaccurate 24 hr urine collection
Creatinine
Advantages Disadvantages
Endogenous
Analytes Used For Calculating GFR
inaccurate 24 hr urine collection
• Gold standard for measurement of GFR
• Cumbersome
• Expensive
• Limited availability
• Usually limited to clinical research
• Close correlation with inulinclearance
• Widespread availability
• Non-radiolabeled analyte
• Timed clearance
Inulin
Iothalamate
EndogenousExogenous
Emory Transplant Center GFR Assessment
Pt Void (UO) Pt Void (U1)/Plasma (P2)Pt Void (UE)/Plasma (P1)
Clinical Relevance
0 min
300mg Iothalamate SubQ
110 min
Iothalamate U1 (Urine) (µg/mL) X flow (mL/min)Iothalamate Avg P1 & P2 (Plasma) (µg/mL)
GFR =
65 min5 min
Iothalamate MRM
Iothalamate
Iohexol
Iohexol
Product Ion #2
Product Ion #1
Time (min) Time (min)
IohexolIothalamate
Product Ion #2
Product Ion #1
H+
NH4+
Na+
Ion Suppression
Methanol Supernatant Dried Ext in Mobile Phase A
SampleIothalamate
Area SampleIothalamate
Area
Exp #1 Exp #2
Sample Area Sample Area
Mobile Phase 101905 Mobile Phase 69508
Plasma 1 31941 Plasma 1 67397
Plasma 2 30611 Plasma 2 59226
Plasma 3 32959 Plasma 3 64639
Suppression 69% Suppression 8%
Deming Regression Bland-Altman
Accuracy
y = 1.07x + 1.80R2 = 0.96
y = 1.09x + 0.19R2 = 0.73
Linear Regression Bland-Altman
y = 1.06x – 11.25R2 = 0.80
GFR
Total Error = Bias + (1.96 X SD)
Total Error = 5.85 + (1.96 X 6.2)
Total Error = 18%
BiasMDL = (mx + b) – xMDL
BiasMDL = (mx + b) – 90 mL/min
BiasMDL = 5.85
A UPLC LC-MS/MS Assays for the Newer Antidepressants,
Antipsychotics, and their Active MetabolitesMetabolites
Why Monitor?
• Check Adherence
• Differential Metabolism
• Rough Therapeutic Ranges• Rough Therapeutic Ranges
• To Check Clearance of Drug When Switching to
Another or in Special Populations
• Suspect Serotonin Syndrome
• Suspect Overdose
Types of “Newer” Antidepressants
• Selective Serotonin Reuptake Inhibitors (SSRIs)
– Citalopram (Celexa)
– Escitalopram (Lexapro)
– Fluoxetine (Prozac)
– Fluvoxamine (Luvox)
– Paroxetine (Paxil)
– Sertraline (Zoloft)
• Serotonin-norepinephrine reuptake inhibitors (SNRIs)
– Duloxetine (Cymbalta)
– Milnacipram (Ixel)
– Venlafaxine (Effexor)
– des-Venlafexine (Pristiq)
Types of “Newer” Antidepressants
• Noradrenergic & specific serotonergic antidepressants (NaSSAs)
– Mianserin (Tolvon)
– Mirtazapine (Remeron, Avanza, Zispin)
• Norepinephrine reuptake inhibitors (NRIs) • Norepinephrine reuptake inhibitors (NRIs)
– Atomoxetine (Strattera)
– Mazindol (Mazanor, Sanorex)
– Reboxetine (Edronax)
– Viloxazine (Vivalan)
• Norepinephrine-dopamine reuptake inhibitors (NDRIs)
– Bupropion (Wellbutrin, Zyban)
2nd Generation Antipsychotics
Antipsychotic Drugs Trade Name
Olanzapine (+active metab) Zyprexa
Risperidone Risperdal
9-OH Risperidone Paliperidone9-OH Risperidone Paliperidone
Ziprasadone Geodon
Clozapine(+active metab) Clozaril
Haloperidol Haldol
Quetiapine Seroquel
Aripiperazole (+active metab) Abilify
Atomoxetine(+active metab) Strattera
Objectives
• To develop LC-MS/MS methods to measure serum levels of the newer ADs and APs using “off the shelf reagents”
• Methods must also measure active • Methods must also measure active metabolites of these compounds
• Method must be robust and cost effective versus sending to a reference laboratory
Sample Extraction
• 100 µL Sample, QC, or Standard
• (AD) = 10 µL Mobile Phase A
(AP) = 50 uL 0.4M ZnSO4 , sit 2 mins
• 200 µL Internal Standard (in MEOH)
• Vortex 1 min, let stand 5 mins
• Centrifuge at 14,000xG for 5 mins
• Transfer supernatant to sample vial
• Inject 5 µL of extract
Compound Parent Ion Daughter Ion Cone (V) Collision (eV) Dwell RT(min)
des-Mirtazapine 252.13 195.10 43.00 18.00 0.01 1.17
ODV 264.00 106.70 30.00 37.00 0.01 0.95
Mirtazapine 266.00 194.70 39.00 23.00 0.01 1.15
d3-Mirt 269.00 194.70 39.00 23.00 0.01 01.14
Venlafaxine 278.20 120.80 30.00 35.00 0.01 1.74
d6-Ven 284.00 120.80 30.00 35.00 0.01 1.72
des-Sertraline 291.80 158.50 17.00 25.00 0.01 2.75
des-Fluoxetine 295.90 133.90 20.00 5.00 0.01 2.58
MRM - ADs
des-Fluoxetine 295.90 133.90 20.00 5.00 0.01 2.58
Douloxetine 298.00 153.70 18.00 6.00 0.01 2.43
d6-des-Fluox 301.80 139.80 20.00 5.00 0.01 2.57
Sertraline 305.90 158.50 20.00 25.00 0.01 2.69
d3-Sert 308.90 158.50 20.00 25.00 0.01 2.69
Fluoxetine 310.20 148.10 30.00 10.00 0.01 2.56
Reboxetine 314.00 175.60 31.00 13.00 0.01 2.18
Fluvoxamine 319.20 258.20 30.00 10.00 0.01 2.52
Citalopram 325.10 108.90 40.00 25.00 0.01 1.94
Paroxetine 330.00 69.80 30.00 30.00 0.01 2.34
d6-Parox 336.00 75.80 35.00 30.00 0.01 2.34
Tuned to Max Sensitivity for des-Sertraline
Compound Parent Ion Daughter Ion
20
ionCone (V) Collision (eV) Dwell RT
(min)
d8-N-desmethyl-Olanzapine
307.00 260.20 212.70 40.00 35.00 0.01 1.53
N-desmethyl-Olanzapine 299.00 255.80 212.70 38.00 26.00 0.01 1.54
Olanzapine 313.00 255.80 197.70 34.00 25.00 0.01 1.57
4-OH Atomoxetine 272.00 44.00 21.00 14.00 0.01 2.15
9-OH-Ripseridone 427.00 206.90 109.90 45.00 31.00 0.01 2.14
d3,13C2-Risperidone 415.50 192.90 109.90 47.00 31.00 0.01 2.20
Risperidone 411.10 190.90 109.90 47.00 31.00 0.01 2.20
d8-Ziprasidone 421.00 193.80 46.00 32.00 0.01 2.49
Ziprasidone 413.00 193.80 46.00 32.00 0.01 2.50
MRM MRM -- APsAPs
Ziprasidone 413.00 193.80 46.00 32.00 0.01 2.50
nor-Clozapine 313.00 269.90 40.00 25.00 0.01 2.55
d8-Clozapine 335.00 274.70 40.00 25.00 0.01 2.58
Clozapine 327.00 270.00 38.00 25.00 0.01 2.59
d4-Haloperidol 380.00 168.90 126.90 40.00 26.00 0.01 2.64
Haloperidol 375.90 164.90 122.80 40.00 26.00 0.01 2.65
Quetiapine 384.00 252.80 45.00 27.00 0.01 2.64
d7-Atomoxetine 263.10 43.90 19.00 13.00 0.01 2.72
Atomoxetine 256.00 43.90 19.00 13.00 0.01 2.72
dehydro-Aripiperazole 446.00 284.80 36.00 25.00 0.01 2.70
d8-Aripiperazole 456.00 292.80 38.00 38.00 0.01 2.71
Aripiperazole 448.00 284.80 175.90 38.00 30.00 0.01 2.72
Tuned to Max Sensitivity for n--desmethyl-Olanzapine
ADsADs -- 100 100 ngng//mLmL Std (TIC)Std (TIC)
MIRTs Paroxs
Fluv & Fluox
ODV
Vs+C
Rebox
Duolox
Fluv & Fluox
des-Fluoxs
Serts
APsAPs -- 100 100 ngng//mLmL STD (TIC)STD (TIC)
Risp
Olanz’s
OH-Atomox + OH-Risp
Zip
Cloz’s
Hal + Quet
Atomox + Aripip’s
ImprecisionIntra-assay:ADs ADs - 5 to 10% at 75 & 300 ng/mL (n=10)APs APs - 4 to 11% at 5/100 & 15/350ng/ml (n=10)
Interassay:
ADsADs - 7 to 12% at 75 & 300 ng/mL (n=10)APs APs - 3 to 12% at 5/100 & 15/350 ng/mL (n=13)
Linearity
Assays PerformanceAssays Performance
Linearity ADsADs – 0.2 to 2000 ng/mL (LOD=0.05, des-Sert = 0.1)APs APs – 0.2 to 2000 ng/mL (LOD=0.05 ng/mL)
Matrix Effects (Matuszewski, et al 2003)ADs ADs - 11 (reboxetine) to -3% (Venlafexine)APsAPs - <4% for all compounds
Absolute RecoveryADsADs – 75 to 114% from 5 to 550 ng/mLAPsAPs – 84 to 108% from 2.3 to 228 ng/mL
Citalopram Assay Comparison
y = 0.7883x - 0.3088
R2 = 0.7537
50
100
150
LC/MS/MS
(ng/m
L)
Assay ComparisonsAssay ComparisonsEmory HPLCEmory HPLC--UV vs LC/MS/MSUV vs LC/MS/MS
0
0 50 100 150HPLC-UV
(ng/mL)
Paroxetine Comparison
y = 0.9341x - 21.943
R2 = 0.9689
0
100
200
300
400
500
600
700
800
0 200 400 600 800
HPLC-UV
LC/MS/MS
Flouxetine
y = 0.7021x + 13.284
R2 = 0.7917
0
100
200
300
0 50 100 150 200 250 300
UK - All Methods Mean
Emory-LC/MS/MS
des-Fluoxetine
y = 0.6895x + 7.9303
R2 = 0. 6832
0
50
100
150
200
250
300
0 50 100 150 200 250 300
U K - A l l M e t h o d s M e a n
Sertraline
y = 1. 18 9 8 x - 4 . 0 8 8 2
R2 = 0 . 9 9 3 7
9 0
12 0
15 0
des-Sertraline
y = 1.0415x + 0.3831
R2 = 0. 9312
200
250
300
Assay Comparisons Assay Comparisons -- UKEQASUKEQAS
0
3 0
6 0
0 3 0 6 0 9 0 12 0 15 0
U K - A ll M e t hods M e a n
0
50
100
150
0 50 100 150 200 250 300
U K - A l l M e t h o d s M e a n
Venlafexine
y = 1.1114x + 50.656
R2 = 0.8056
0
100
200
300
400
500
0 100 200 300 400 500
U K - A l l M e t h o d s M e a n
ODV
y = 0.9163x + 28. 79
R2 = 0.7573
0
100
200
300
400
500
0 100 200 300 400 500
U K - A l l M e t h od s M e a n
Clinical Use
• 2010 VolumesParoxetine – 15
Fluoxetine – 20
Citalopam – 18
Considerations:
1. AD and AP assays are essentially
the same for the technologist
2. Reagents, Standards , and
controls are very stable at -80CCitalopam – 18
Venlafaxine - 12
Sertraline – 14
Olanzapine – 15
Risperidone - 18
Clozapine – 52
Quetiapine – 11
Aripiperazole - 5
• Total = 180/yr
controls are very stable at -80C
3. No stat turnaround requirements
4. Can be used to fill down time on
LC-MS
Small runs can be cost effective !
Lab Test of the Decade
800
1000
1200
Vitamin D deficiency
0
200
400
600
20
00
20
01
20
02
20
03
20
04
20
05
20
06
20
07
20
08
20
09
20
10
PubMed Search
Lab Test of the DecadeEmory Medical Laboratories
Financial Justification
• EML:
– Vitamin D biggest send-out test
– A new LC–MS/MS instrument was purchased for
vitamin D testing:
• Waters Xevo TQ MS• Waters Xevo TQ MS
• 28716 tests @ a cost of $13.05
• Estimated cost by mass spec:
– Reagents/calibrators/ standards/ labor : $4.50
• Estimated savings: $8.55/test
– ~ $200K/yr (after factoring in maintenance contract)
Acquisition of Materials
• Endogenous compounds:
– Stripped sera vs standards prepared in BSA
• Golden West Biologics
– Vitamin D 25-Hydroxy, D2 & D3 Total < 1 ng/ml Trigylcerides &
Cholesterol < 10 mg/dlCholesterol < 10 mg/dl
– Vitamin D 25-Hydroxy, D2 & D3 Total < 1 ng/ml, Triglycerides
& Cholesterol normal range
• 4% BSA
• Calibrators & QC materials were
purchased from Chromsystems
• Internal Standard: D6 25-OH D3 (Cerillient)
Sample Extraction
• To 300 µL of serum or plasma, add: – 20 µL of internal std (D6 25-OH D3, 250 ng/mL in
EtOH),
– 150 µL 0.2 mol/L ZnSO4, and vortex.
– Add 300 µL MeOH, – Add 300 µL MeOH,
– 750 µL hexane, vortex and centrifuge.
• Transfer 400 µL of the hexane layer to a glass tube and dry under a stream of N2.
• Reconstitute with 75 µL mobile phase A and transfer to LC vial.
• Inject 20 µL.
Chromatographic Separation
D3: 3.18 min
401.3 > 159.2
D6: 3.18 min
407.2 > 159.2
D2: 3.26 min
413.4 > 355.3
What’s Next ??
Proteomics
The systematic analysis and documentation of
proteins in biological samples with a focus on
state-related expression of proteins.”state-related expression of proteins.”
Proteomics is the study of multiprotein systems, in which the focus is on the interplay of multiple, distinct proteins in their roles as part of a larger system or network (systems biology).
Challenges
Lancet, 2002, 359:572-577
Technical ChallengesAnalytical:
– Must span up to 10 orders of magnitude in concentration
– Be able to detect multiple forms of the same analyte (PTMs, isoforms, etc)
– Lack of a generally applicable technique for – Lack of a generally applicable technique for protein quantitation
– Limited throughput of todays proteomic platforms
– Problems with high MW, basic, or hydrophobic proteins
Technical Challenges
Bioinformatics:
• Analyses produce a huge amount of data
• Must have comprehensive databases to get accurate sequence information and accurate sequence information and identification.
• No agreed upon algorithms for standardization of analyses
Challenges of Biomarker Discovery in
Plasma
• Currently there is no single comprehensive proteomics platform for plasma.
• Multi-dimensional fractionation is crucial to penetrate deeper into the abundance distributionpenetrate deeper into the abundance distribution
• Where possible discovery efforts should center on tissues or non-plasma fluids where they occur at higher concentrations
• “Ultimately however we need to measure biomarkers in plasma to be most effective”
L. Anderson, Plasma Protein Institute
The way forward
• Proteomics currently is a biomarker discovery tool which promises to revolutionize our thinking about health and disease.
• This technology may someday provide disease specific panels to aid diagnosis and treatment.panels to aid diagnosis and treatment.
• Many barriers must be overcome before these assays reach the clinical laboratory.
• However, just as with small molecules, these new techniques can be employed incrementally to solve some of our knotty clinical problems.
Thyroglobulin (Tg)
• A 660 Kd dimeric thyroid protein which forms the backbone for thyroid hormone production
• Serum Tg is used in conjunction with imaging to manage patient treatment after removal of thyroid carcinoma.thyroid carcinoma.
• Approximately 10 to 25% of patients have anti-Tg antibodies which potentially can interfere with the immunoassays commonly used to measure Tg
• Commercial Tg immunoassays lack concordance across platforms most likely due to posttranslational modifications in vivo.
1.1.1.1. Method uses stable isotope standards and capture by antiMethod uses stable isotope standards and capture by antiMethod uses stable isotope standards and capture by antiMethod uses stable isotope standards and capture by anti----1.1.1.1. Method uses stable isotope standards and capture by antiMethod uses stable isotope standards and capture by antiMethod uses stable isotope standards and capture by antiMethod uses stable isotope standards and capture by anti----peptide antibodies (SISCAPA)peptide antibodies (SISCAPA)peptide antibodies (SISCAPA)peptide antibodies (SISCAPA)
2.2.2.2. Three digest peptides are used to assure specificityThree digest peptides are used to assure specificityThree digest peptides are used to assure specificityThree digest peptides are used to assure specificity3.3.3.3. Analytical sensitivity of 2.6 Analytical sensitivity of 2.6 Analytical sensitivity of 2.6 Analytical sensitivity of 2.6 ngngngng/ml approaches that of /ml approaches that of /ml approaches that of /ml approaches that of
immunoassay (1.0ng/immunoassay (1.0ng/immunoassay (1.0ng/immunoassay (1.0ng/mLmLmLmL))))4.4.4.4. No interference from antiNo interference from antiNo interference from antiNo interference from anti----TgTgTgTg antibodiesantibodiesantibodiesantibodies
Comparison to ImmunoassayComparison to Immunoassay
Other Targets of Opportunity• Β-hCG (Beta human chorionic gonadotropin)
• Sex Hormone Binding Globulin
• Lipoproteins (LPa. Apo-B, B-100, Apo A1, etc)
• C-reactive protein (CRP)
• D-dimers
• α -acid glycoprotein • α1-acid glycoprotein
• ACTH
• Glycated albumin
• Βrain-Natriuretic peptide (BNP)
• Ferritin
• α-Fetoprotein
• And many many more……
1. Enables an unbiased identification of microorganisms.
2. Can be used for gram-positive and gram-negative bacteria, yeast and multicellular fungi without presumptions or pretesting.
3. Applications from clinical microbiology, food, feed safety and analysis
MALDI Biotyper: The next generation microbial identification system for the 21st century
Microbiology ?
Microbial identification system based on bench top microflexMALDI-TOF mass spectrometer
•Unbiased identification of bacteria, yeasts and multicellularfungi•Cost-effective sample preparation•High resolution identification down to species level in one step•Analysis of mixed cultures•Interfaces for LIMS integration
J.Banoub (ed.) Detection of Biological Agents for the Prevention of Bioterrorism,2010, Springer Sci
J.Banoub (ed.) Detection of Biological Agents for the Prevention of Bioterrorism, 2010, Springer Sci
Status of LC-MS/MS Assays at EML
Today:
Clinically in Use
CyclosporineRapamycinTacrolimusMycophenolic acidBusulfanAntidepressants (14)
In Development
25 (OH) Vitamin DAntidepressants (14)Antipsychotics (12)
Research
ArgatrobanLenalidomideLevamisoleBile AcidsIodothalamateFlumazenil
25 (OH) Vitamin DEverolimusGlucocorticoidsTestosteroneMetanephrines
� High specificity
� Multiplexing possible
� High sensitivity (femtomolar LODs) and accuracy
� Wide linear range
Throughput
AutomationAccuracy
Selectivity
Advantages of MS in the Clinical Advantages of MS in the Clinical LaboratoryLaboratory
�
� Limited sample prep with short chromatographic run-times
� Low sample volumes –pediatrics
� Alternative Matrices – blood spots, saliva, hair
Automation
Ease of UseSensitivity
Accuracy
Requirements of Clinical Laboratory Methodology
� Automation (including sample prep)
� Ease of Use - (autotunes & autocals), smart systems
� LIS connectivity
Throughput
AutomationAccuracy
Selectivity
Requirements for MS to gain wide acceptance Requirements for MS to gain wide acceptance in Clinical Laboratoriesin Clinical Laboratories
� Low carryover
� 24 hr service
� Manufacturer supported procedures and reference ranges
� Cost Effectiveness
Automation
Ease of UseSensitivity
Accuracy
Requirements of Clinical Laboratory Methodology
Today I’ve shown you:• A new instrument to
understand….
• A few new concepts……
• A few new things to watch
out for……….
• A glimpse of the future……
Ultimately to provide more accurate analyses And better patient care
Remember all instruments are simply tools
AcknowledgementsAcknowledgements
ECTRL Laboratory Staff Clinical Chemistry FellowsPatricia Scott-Harrell, B.S. Marion Snyder, Ph.D.Bailey Glover, M.D. Ross Molinaro, Ph.D.Clay Ramsey, B.A. Charbel AbuDiwan, Ph.DLisa Maxwell, RN
EML Special Chemistry StaffWilla Zhang, MSCora Tomblin, MT (ASCP)Tom Tuten, MS
http://www.pathology.emory.edu/ECTRL/
Tom Tuten, MS
QUESTIONS ???
Methodology
• EML had analyzed CsA previously using the Abbott monoclonal immunoassay on the TDx/Flx in our Core Laboratory (“in by 10am, results by 3pm”).
• Therapeutic Range: 50 to 400 ng/mL.
• A semi-automated FPIA assay.
• Cross-reactivity with metabolites: AM1 = 9.3%, AM9 = 23.5%.(Steimer
W, 1999, Clin Chem, 45:371-381)
• The cross-reactivity of the CsA metabolites in the Abbott monoclonal assay matches closely with their pharmacological potency in the MLC assay. (Murthy et al, 1998, Clin Biochem, 31:159-163)
Hamwi, et al, Cyclosporine Metabolism in Patients after
Kidney, BoneMarrow, Heart-Lung, and Liver Transplants in Early and late
Posttransplant Periods, Am J Clin Pathol, 114:536-543, 2000
Transplant Type
White bars = Early period (≤ 3 months)
Black bars = Late period (>3 months)
Transplant TypeA=KidneyB=Bone MarrowC=Heart-LungD=Liver
Hamwi, et al, Cyclosporine Metabolism in Patients after
Kidney, BoneMarrow, Heart-Lung, and Liver Transplants in Early and late
Posttransplant Periods, Am J Clin Pathol, 114:536-543, 2000
Transplant TypeA=KidneyB=Bone MarrowC=Heart-LungD=Liver
White bars = Early period (≤ 3 months)
Black bars = Late period (>3 months)
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