mass spectrometry as the premier analytical tool in drug discovery and drug development
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Mass Spectrometry as the Premier Analytical Tool in Drug Discovery and Drug Development. Walter Korfmacher Exploratory Drug Metabolism Merck Research Laboratories Kenilworth, NJ USA. Outline. New Drug Discovery Challenges Mass Spectrometry Basics - PowerPoint PPT PresentationTRANSCRIPT
1October 14, 2010 GBMSDG Talk
Mass Spectrometry as the Mass Spectrometry as the Premier Analytical Tool in Premier Analytical Tool in Drug Discovery and Drug Drug Discovery and Drug
DevelopmentDevelopment
Walter KorfmacherWalter Korfmacher
Exploratory Drug Metabolism Exploratory Drug Metabolism Merck Research LaboratoriesMerck Research Laboratories
Kenilworth, NJ USAKenilworth, NJ USA
2October 14, 2010 GBMSDG Talk
OutlineOutline New Drug Discovery Challenges New Drug Discovery Challenges Mass Spectrometry BasicsMass Spectrometry Basics Selected In vitro Drug Selected In vitro Drug
Metabolism ApplicationsMetabolism Applications Selected In vivo Drug Selected In vivo Drug
Metabolism ApplicationsMetabolism Applications Metabolite ID ApplicationsMetabolite ID Applications MS Imaging ApplicationsMS Imaging Applications ConclusionsConclusions
3
Topics Not CoveredTopics Not Covered
ProteomicsProteomics Biomarker discovery or assayBiomarker discovery or assay MetabolomicsMetabolomics High Throughput ScreeningHigh Throughput Screening
October 14, 2010 GBMSDG Talk
4GBMSDG Talk
Drug Discovery:Drug Discovery:From Library to MarketFrom Library to Market
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Compound Libraries
Lead Selection and Optimization
Stages of Discovery and Development
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Early DiscoveryLead Optimization Safety Testing Clinical
TestingFDA Approval
October 14, 2010
5October 14, 2010GBMSDG Talk
NEW DRUG DISCOVERY PIPELINE
Chemistry
Biology--HTS for Receptor Activity
In-vitro Stability Screen
In-vitro Absorption Screen
P450 Enzyme Inhibition Screen
CARRS Oral PK Screen
Rat IV / PO PK
Dog and Monkey IV / PO PK
Rising Dose and Multiple Dose Studies and Safety Screens
Drugs into Development
Metabolite ID
DMPK
Lead Optimization
6October 14, 2010 GBMSDG Talk
The ChallengeThe Challenge
How to deal with the multiple How to deal with the multiple compounds at multiple stages in the compounds at multiple stages in the drug discovery/drug development drug discovery/drug development pipeline.pipeline.
7October 14, 2010 GBMSDG Talk
The SolutionThe Solution
LC-MS and LC-MS/MSLC-MS and LC-MS/MS
8October 14, 2010 GBMSDG Talk
Why use Mass Why use Mass Spectrometry?Spectrometry?
Specificity!Specificity! Non-specific techniques, Non-specific techniques, such as UV and fluorescence, are unable such as UV and fluorescence, are unable to provide proof of the analyte identityto provide proof of the analyte identity
Ease of Use!Ease of Use! Modern mass Modern mass spectrometry software interfaces are spectrometry software interfaces are easy to useeasy to use
Versatility!Versatility! MS is both a qualitative MS is both a qualitative and quantitative techniqueand quantitative technique
9October 14, 2010 GBMSDG Talk
The ChallengeThe Challenge
Choosing the right tool for the task :Choosing the right tool for the task :
MS Toolbox
Hammer?
OR
Wrench?
10October 14, 2010 GBMSDG Talk
Common MS Tools
11October 14, 2010 GBMSDG Talk
What is LC-MS?What is LC-MS?Liquid Chromatography Coupled to a Liquid Chromatography Coupled to a Mass SpectrometerMass Spectrometer(In this case the Mass Spectrometer is a (In this case the Mass Spectrometer is a Single Quadrupole instrument)Single Quadrupole instrument)
HPLC Column Ion source Mass analyzer Detector
APCI or ESI
12October 14, 2010 GBMSDG Talk
The Challenge—Compound The Challenge—Compound SynthesisSynthesis
At a big Pharma site, one might find At a big Pharma site, one might find hundreds of medicinal chemists who hundreds of medicinal chemists who might produce 200-1000 new might produce 200-1000 new compounds each week. These have compounds each week. These have to be assayed.to be assayed.
13October 14, 2010 GBMSDG Talk
The Solution—LC-MSThe Solution—LC-MS
• The LC-MS system based on a single quadrupole MS is a very useful tool for medicinal chemists who want to know if their synthesis is working correctly—did they make the right compound? Often this is set up
as an open access tool. The chemists set up the run and get results within 24 hours.
14October 14, 2010 GBMSDG Talk
The Challenge—Lead The Challenge—Lead Optimization In Vitro Optimization In Vitro
ScreeningScreening At a big Pharma site, one might get At a big Pharma site, one might get
100-200 new compounds each week 100-200 new compounds each week that have to be screened in various that have to be screened in various in vitro assays. These have to be in vitro assays. These have to be assayed separately for each screen.assayed separately for each screen.
15October 14, 2010 GBMSDG Talk
The Solution—LC-MS/MSThe Solution—LC-MS/MS
• The LC-MS/MS system based on a triple quadrupole MS system is the tool of choice for most quantitative discovery bioanalytical applications.
The application of this tool varies with the screen.
16October 14, 2010 GBMSDG Talk
What is LC-MS/MS aka What is LC-MS/MS aka Triple Quadrupole Triple Quadrupole
Technology?Technology?Liquid Chromatography Coupled to a Liquid Chromatography Coupled to a TandemTandem Mass Spectrometer Mass Spectrometer (In this (In this case the Mass Spectrometer is a Triple case the Mass Spectrometer is a Triple Quadrupole instrument)Quadrupole instrument)
Q1 Q2 Q3
HPLC Column Ion source Mass analyzer Detector
17October 14, 2010 GBMSDG Talk
Quantitation Gold Standard:Quantitation Gold Standard:MS/MS – Selected Reaction MS/MS – Selected Reaction
Monitoring (SRM)*Monitoring (SRM)*
Select Fragment Selectprecursor ion in Q1 precursor ion in Q2 product ion in Q3
e.g. m/z 216 (Collision Cell) e.g. m/z 174
* Often referred to as Multiple Reaction Monitoring (MRM)
18October 14, 2010 GBMSDG Talk
Effects of Stages of Analysis Effects of Stages of Analysis on on
Signal, Noise, and Signal-to-Signal, Noise, and Signal-to-NoiseNoise
1 2 3 4
S/NSignal
Noise
Stages of Analysis
LC LC-MS LC-MS/MS ?
Important Concept!!
19October 14, 2010 GBMSDG Talk
RT: 0.00 - 5.02
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2.632.262.17
2.001.81 4.62
1.371.010.42 0.610.22
2.83
2.68 3.682.95 3.57 3.75 4.382.56 4.472.32 4.721.981.010.32 1.30 1.900.50
NL:1.35E7
TIC MS plasma01
NL:3.81E5
m/z= 373.0-375.0 MS plasma01
LC-TIC
LC-MS
20October 14, 2010 GBMSDG Talk
RT: 0.00 - 5.02
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2.74 3.89 3.933.01 4.34 4.563.801.10 3.602.612.092.041.141.050.690.42
NL:3.22E3
TIC F: + c APCI SRM ms2 [email protected] [ 120.70-121.30] MS npy086G_004
10 ng/ml
LC-MS/MS
21October 14, 2010 GBMSDG Talk
Discovery In Vitro Discovery In Vitro ScreeningScreening
P450 Assay--Enzyme Inhibition P450 Assay--Enzyme Inhibition Screen Screen
Caco-2 cells—Absorption ScreenCaco-2 cells—Absorption Screen Liver Microsomes/Hepatocytes--Liver Microsomes/Hepatocytes--
Metabolic Stability ScreenMetabolic Stability Screen Plasma Protein BindingPlasma Protein BindingEach In Vitro Assay Uses LC-MS/MS for the analytical step (typically a triple quadrupole MS system).
22October 14, 2010 GBMSDG Talk
High Throughput CYP High Throughput CYP Inhibition Assay ExampleInhibition Assay Example• Generic LC-MS/MS methodGeneric LC-MS/MS method
• 1 minute gradient1 minute gradient• Monitors 3 substrates in a single LC-MS/MS Monitors 3 substrates in a single LC-MS/MS
runrun
• Template is used for creating sample list Template is used for creating sample list
• Automatic results calculation and import Automatic results calculation and import into Activity Baseinto Activity Base
23October 14, 2010 GBMSDG Talk
Evaluate direct and mechanism-based inhibitors for P450 enzymes
(3A4, 2D6, 2C9) to assess potential for drug-drug interactions.
Purpose:
In Vitro Evaluation of CYP Inhibition
incubation cocktail ( 3A4, 2D6, 2C9)
detection LC-MS-MS
P450 source human liver
substrates testosterone Dextromethorphan Tolbutamine
products 6-hydroxytestosterone dextrophan 4-hydroxytobutamide
3A4 2D6 2C9
Method:
incubation cocktail
detection LC-MS-MS
P450 source human liver
substrates testosterone Dextromethorphan Tolbutamine
products 6-hydroxytestosterone dextrophan 4-hydroxytobutamide
3A4 2D6 2C9
incubation
detection LC-MS-MS
P450 source human liver microsomes
substrates testosterone Dextromethorphan Tolbutamine
products 6-hydroxytestosterone dextrophan 4-hydroxytobutamide
3A4 2D6 2C9
Method:
24October 14, 2010 GBMSDG Talk
In VitroIn Vitro Evaluation of CYP Evaluation of CYP InhibitionInhibition
Stock solution
from CDCSerial dilution
incubation/pre-incubation
1. Coincunation
2. Coincubation
3. Preincubation
4. Preincubation
•three concentrations/cpd: 20 M, 2 M and 0.2 M
•duplicates/each conc.
•30 compounds/set
25October 14, 2010 GBMSDG Talk
LC-MS/MS for p450 LC-MS/MS for p450 Inhibition Screen-Inhibition Screen-Run time Run time
is less than 1 minuteis less than 1 minute
XIC of +MRM (4 pairs): 305.2/269.4 amu from Sample 27 (616005) of 090616 3in1 Hui 4 plates.wiff (Heated Nebulizer), Smoothed Max. 1.3e5 cps.
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9Time, min
0.00
5.00e4
1.00e5
1.29e5
In
te
ns
it
y,
c
ps
0.76
XIC of +MRM (4 pairs): 258.1/157.1 amu from Sample 27 (616005) of 090616 3in1 Hui 4 plates.wiff (Heated Nebulizer), Smoothed Max. 3.8e5 cps.
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9Time, min
0.0
1.0e5
2.0e5
3.0e5
3.8e5
In
te
ns
it
y,
c
ps
0.61
XIC of +MRM (4 pairs): 347.2/121.3 amu from Sample 27 (616005) of 090616 3in1 Hui 4 plates.wiff (Heated Nebulizer), Smoothed Max. 2.0e5 cps.
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9Time, min
0.0
5.0e4
1.0e5
1.5e5
2.0e5
In
te
ns
it
y,
c
ps
0.81
XIC of +MRM (4 pairs): 287.0/170.9 amu from Sample 27 (616005) of 090616 3in1 Hui 4 plates.wiff (Heated Nebulizer), Smoothed Max. 7.1e4 cps.
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9Time, min
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4.0e4
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0.69
0.74
Substrate for CYP3A4, 6-hydroxytestosterone
Substrate for CYP2D6, dextrophan
Internal Standard
Substrate for CYP2C9, 4-hydroxytolbutamide
m/z 305 269
m/z 258 157
m/z 287 171
m/z 347 121
26October 14, 2010 GBMSDG Talk
CYP Inhibition Screen CYP Inhibition Screen ThroughputThroughput
Throughput: ~ 150 compounds /week, ~ 7000 samples/week
Analytical cycle-time: 48 hr from delivery to results
•NOTE: The advantage for this assay is that it is the same regardless of the test compound—no compound method development needed.
27October 14, 2010 GBMSDG Talk
CYP Inhibition Screen CYP Inhibition Screen ReviewReview
28October 14, 2010 GBMSDG Talk
The Challenge— Metabolic The Challenge— Metabolic Stability ScreeningStability Screening
At a big Pharma site, one might get At a big Pharma site, one might get 100-200 new compounds each week 100-200 new compounds each week that have to be screened for that have to be screened for metabolic stability. metabolic stability.
The challenge is that LC-MS/MS The challenge is that LC-MS/MS methods have to be developed for methods have to be developed for each compound.each compound.
29October 14, 2010 GBMSDG Talk
The Solution—LC-MS/MS + The Solution—LC-MS/MS + Software + HardwareSoftware + Hardware
• Use a generic HPLC method. This works for 80-90% of the compounds.
• Use a vendor-supplied software tool for automated MS/MS method development, e.g.:
• QuickQuan™ (Thermo-Fisher)QuickQuan™ (Thermo-Fisher)
• QuanOptimise™ (Waters-Micromass)QuanOptimise™ (Waters-Micromass)
• DiscoveryQuant™ (AB-Sciex)DiscoveryQuant™ (AB-Sciex)
• OptimizerOptimizerTM TM (Agilent)(Agilent)
• Use robots for automated sample handlingUse robots for automated sample handling
30October 14, 2010 GBMSDG Talk
Metabolic Stability Assay Metabolic Stability Assay ExampleExample
31October 14, 2010 GBMSDG Talk
Metabolic Stability AssayMetabolic Stability Assay
Layout shows how a robotic liquid handler can be used to perform the incubation and sample preparation steps in a metabolic stability assay.
32October 14, 2010 GBMSDG Talk
Metabolic Stability AssayMetabolic Stability Assay
Scheme shows how a well organized system is needed to provide high throughput metabolic stability data.
33October 14, 2010 GBMSDG Talk
In Vivo AssaysIn Vivo Assays Various types of in vivo assays are Various types of in vivo assays are
performed as part of new drug discovery performed as part of new drug discovery and developmentand development
The goal may be to understand absorption The goal may be to understand absorption (A), distribution (D), metabolism (M), or (A), distribution (D), metabolism (M), or excretion (E) properties of a compoundexcretion (E) properties of a compound
The goal may be to get pharmacokinetic The goal may be to get pharmacokinetic (PK) information on a compound(PK) information on a compound
34ASMS 2010
ADME-PK StudiesADME-PK Studies
Brain-- D
Drug Levels—
LC-MS/MS
MS Image—
MALDI-MS/MS
Liver— D
Drug Levels—
LC-MS/MS
Plasma— A
Drug Levels—
LC-MS/MS
PK Parameters
Dose NCE (Drug) PO/IV
Ref: “Using Mass Spectrometry for Drug Metabolism Studies”
W. Korfmacher, ed., CRC Press, 2005.
35October 14, 2010 GBMSDG Talk
The Challenge— In Vivo PK The Challenge— In Vivo PK ScreeningScreening
At a big Pharma site, one might get At a big Pharma site, one might get 50 - 100 new compounds each week 50 - 100 new compounds each week that have to be screened for in vivo that have to be screened for in vivo PK. PK.
The challenge is that LC-MS/MS The challenge is that LC-MS/MS methods have to be developed for methods have to be developed for each compound.each compound.
36October 14, 2010 GBMSDG Talk
The Solution—LC-MS/MS + The Solution—LC-MS/MS + Software + PlanningSoftware + Planning
• Use a generic HPLC method. This works for 80-90% of the compounds.
• Use a vendor-supplied software tool for automated MS/MS method development.
• Use robots for automated sample Use robots for automated sample handling.handling.
• Develop a standard PK screening assay.Develop a standard PK screening assay.
37October 14, 2010 GBMSDG Talk
In Vivo PK ScreeningIn Vivo PK Screening
Source: Drug Discovery Today Volume 13, Numbers 7/8 April 2008Authors: Bo Liu, Jonathan Chang, William P. Gordon, John Isbell, Yingyao Zhou and Tove Tuntland, Department of Pharmacology, Genomics Institute of the Novartis Research Foundation (GNF), San Diego, USA
38October 14, 2010 GBMSDG Talk
PK Screening Example: PK Screening Example: CARRSCARRS
Hours
Dru
g C
on
cen
trat
ion
0 1 2 3 4 5 6
• Each Compound PO Dose + collect 6 time points on two Rats.
• Pool the samples across the two rats 6 samples for assay.
This allows one to see the PK profile out to 6 hours.
Hours
Dru
g C
on
cen
trat
ion
0 1 2 3 4 5 6
• Each Compound PO Dose + collect 6 time points on two Rats.
• Pool the samples across the two rats 6 samples for assay.
This allows one to see the PK profile out to 6 hours.
Provide basic pharmacokinetic information for all rapid rat compounds.• AUC (0-6hr)• Concentration vs Time Profile (0-6 hr)
The Rapid Rat
Throughput: Up to 96 compounds per week
39October 14, 2010 GBMSDG Talk
CARRS ASSAYCARRS ASSAY• Protein Precipitation Sample Preparation• Generic UPLC conditions (1-2 min run time)• Triple Quadrupole MS for assay (Two-point standard curve)• Automated MS method development (QuanOptimize)
6 Samples
Compound 12
34
56
Solvent Blanks
0 standards
25 250 2500
Rat samples Standards Standards25 250 2500
6 Samples
Compound 12
34
56
Solvent Blanks
0 standards
25 250 2500
Rat samples Standards Standards25 250 2500
40October 14, 2010 GBMSDG Talk
Preclinical PK StudiesPreclinical PK Studies Typical study is one compound dosed oral (PO) Typical study is one compound dosed oral (PO)
and IV (Intravenous) in a laboratory animal. The and IV (Intravenous) in a laboratory animal. The goal is to get PK parameters in various goal is to get PK parameters in various preclinical species. preclinical species.
Typically this produces 50-60 plasma samples.Typically this produces 50-60 plasma samples. Sample preparation is protein precipitation.Sample preparation is protein precipitation. A multipoint standard curve is prepared for the A multipoint standard curve is prepared for the
assayassay Analysis is by LC-MS/MS on a triple quadrupole Analysis is by LC-MS/MS on a triple quadrupole
MS/MS system. Usually a generic internal MS/MS system. Usually a generic internal standard is used for the assay.standard is used for the assay.
41October 14, 2010 GBMSDG Talk
Discovery PK Analysis Discovery PK Analysis FlowchartFlowchart
Animal Dosing Protocol
Dose Animal Collect Plasma Samples in a 96-well plate
Robotic Transfer of Aliquots to Assay 96-well Plate
Discovery PK Analysis Flowchart
96-well plate containing plasma
standards and samples
Robotic Sample Preparation
LC-MS/MS system 96-well plate autosampler
Print raw data and assay report
Send results to database
Prepare PK report
Distribute the PK report to the
Discovery Team via E-mail
Manual or Robotic Transfer of Plasma standards to Assay
96-well plate
Weigh Standard and make stock solution
Prepare diluted solutions and plasma standards using robot
Set up Method and enter sample list
The MS/MS instrument is normally a triple quadrupole system
LC-MS/MS test run (PP Sample prep.)
Non-routine options
SRM
#1: Matrix effect
#2: Interference
#3: standard curve linearity
Assay
Enhanced mass resolution
Revised Chromatography
:
LLE
SPE
Samples
FailOK
Rapid MS Method Development ion a Discovery Environment
Xu et al. Anal. Chem.--2005
GBMSDG TalkOctober 14, 2010
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Typical Discovery PK Typical Discovery PK AssayAssay
1 – 10,000 ng/mL
Res
pons
e ra
tio
44October 14, 2010 GBMSDG Talk
Discovery Metabolite IDDiscovery Metabolite ID
Generally this has two components:Generally this has two components:
Lead Optimization Phase--would use Lead Optimization Phase--would use unlabelled compounds and in vitro unlabelled compounds and in vitro samples to look for major routes of samples to look for major routes of metabolism.metabolism.
Pre-Recommendation Phase—Look for Pre-Recommendation Phase—Look for problem metabolites plus in vitro problem metabolites plus in vitro comparison of human to animal comparison of human to animal metabolism.metabolism.
45October 14, 2010 GBMSDG Talk
Discovery Metabolite IDDiscovery Metabolite ID
Mass Spectrometry serves two Mass Spectrometry serves two purposes:purposes:
Finding metabolites-MS systems can be Finding metabolites-MS systems can be used in various ways to find metabolites in used in various ways to find metabolites in multilple biological matrices (e.g., plasma, multilple biological matrices (e.g., plasma, bile, urine).bile, urine).
Structure elucidation—MS systems can be Structure elucidation—MS systems can be used to to obtain partial or complete used to to obtain partial or complete structural identification for the metabolites structural identification for the metabolites
46October 14, 2010 GBMSDG Talk
Triple Quad Scan Triple Quad Scan Functions:Functions:to find metabolitesto find metabolites Neutral Loss ScanNeutral Loss Scan
no prior knowledge of the parent is required—this is no prior knowledge of the parent is required—this is used to look for certain classes of metabolites (e.g., used to look for certain classes of metabolites (e.g., glucuronide, sulfate or glutathione conjugates)glucuronide, sulfate or glutathione conjugates)
Precursor Ion ScanPrecursor Ion Scan only fragmentation pattern of parent is required—may only fragmentation pattern of parent is required—may
find unexpected metabolitesfind unexpected metabolites
SRM/MRMSRM/MRM the fragmentation pattern of parent is used to predict the fragmentation pattern of parent is used to predict
the fragment ions for likely metabolites—some vendors the fragment ions for likely metabolites—some vendors have software tools that make it easy to build a scan sethave software tools that make it easy to build a scan set
47October 14, 2010 GBMSDG Talk
MS Tools
• Triple Quadrupole MS systems are the premier analytical tool for LC-MS quantitative assays. They are also useful for metabolite ID applications
• Q-TOF MS systems are best used for metabolite ID applications and for Imaging MS applications
• QTrap MS systems are excellent tools for quantitative analyses as well as for metabolite ID applications
48October 14, 2010 GBMSDG Talk
QTrap MS Publication QTrap MS Publication In Vivo PK SamplesIn Vivo PK Samples
Simultaneously quantifying parent Simultaneously quantifying parent drugs and screening for metabolites drugs and screening for metabolites in plasma pharmacokinetic samples in plasma pharmacokinetic samples using selected reaction monitoring using selected reaction monitoring information-dependent acquisition information-dependent acquisition on a QTrap instrument:on a QTrap instrument:
Li et al. (Covance), RCMS, 1943, Li et al. (Covance), RCMS, 1943, 2005.2005.
Typical Metabolite Profiling Experiments and Instrumentation
HPLC
Radiometric Flow detector
Mass Spectrometer
Injector
Sample from in vivo or in vitro studies
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0 5 10 15 20 25 30 35 40 45
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10-50%
50-90%
Radioactivity helps to locate the metabolites in the samples
GBMSDG TalkOctober 14, 2010
50October 14, 2010 GBMSDG Talk
Product Ion ScanProduct Ion Scan
Select PrecursorIon
Scan ProductsFragmentation
m1+
m2+
m2+
m2+
Product ion spectrum of a particular compound
A key technique for obtaining structural information.A key technique for obtaining structural information.
51October 14, 2010 GBMSDG Talk
Discovery Metabolite IDDiscovery Metabolite ID
(A) HPLC Radiochromatogram of 14C-Gemfibrozil at 25 mm Incubated in Human Liver Microsomes Fortified with NADPH and UDPGA;
(B) Reconstructed Ion Chromatogram;
(C) Full Scan Mass Spectrum of M1 [M-H]- .
Xia, Y.Q. et al., Use of a quadrupole linear ion trap mass spectrometer in metabolite identification and bioanalysis, Rapid Commun. Mass Spectrom., 17(11), 1137, 2003.
MS/MS spectrum of M1
52October 14, 2010 GBMSDG Talk
What is MIST?What is MIST?MMass Spectrometryass SpectrometryIInvestigatorsnvestigatorsSSecurityecurityTTrustrust MIST will ensure job security MIST will ensure job security
for MS metabolite ID expertsfor MS metabolite ID experts
53October 14, 2010 GBMSDG Talk
What is MIST?What is MIST?MetabolitesMetabolitesIInnSSafetyafetyTTestingesting MIST will ensure job security MIST will ensure job security
for MS metabolite ID expertsfor MS metabolite ID experts
54October 14, 2010 GBMSDG Talk
Key MIST PointsKey MIST Points
1. Human metabolites that can raise a safety concern 1. Human metabolites that can raise a safety concern are those formed at are those formed at greater than 10 percent greater than 10 percent of parent drug’s systemic exposureof parent drug’s systemic exposure at steady state. at steady state.
2. Metabolites identified only in human plasma or 2. Metabolites identified only in human plasma or Metabolites present at disproportionately higher Metabolites present at disproportionately higher levels in humans than in any of the animal test levels in humans than in any of the animal test species should be considered for safety assessment. species should be considered for safety assessment.
Bottom line: Find human metabolites and Bottom line: Find human metabolites and then be sure they are “covered” in the tox then be sure they are “covered” in the tox species.species.
55October 14, 2010 GBMSDG Talk
New MS Tool for Finding New MS Tool for Finding Metabolites: HRMSMetabolites: HRMS
High Resolution Mass Spectrometry High Resolution Mass Spectrometry (HRMS) has become the tool of choice (HRMS) has become the tool of choice for finding metabolites in complex for finding metabolites in complex biological matrices.biological matrices. The improved mass resolution can be used to The improved mass resolution can be used to
differentiate metabolites from endogenous differentiate metabolites from endogenous background background
Software tools can use HRMS to find Software tools can use HRMS to find metabolitesmetabolites
The accurate mass of a detected metabolite The accurate mass of a detected metabolite can help to confirm its identity by leading to can help to confirm its identity by leading to its empirical formulaits empirical formula
56October 14, 2010 GBMSDG Talk
Two HRMS SystemsTwo HRMS Systems
LTQ-Orbitrap
The TOF MS provides mass resolution of 10,000-50,000
The Orbitrap MS provides
mass resolution of 10,000-100,000
57October 14, 2010 GBMSDG Talk
Why High Mass Why High Mass Resolution? TOF-MS Resolution? TOF-MS
of Sidenafil Exampleof Sidenafil ExampleStd 1
Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00
%
0
100
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%
0
100
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%
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%
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%
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FLI_W03052010_raw004 1: TOF MS ES+ 475.216 0.00Da
2.90e31.02
FLI_W03052010_raw004 1: TOF MS ES+ 475.216 0.01Da
2.90e31.02
FLI_W03052010_raw004 1: TOF MS ES+ 475.216 0.10Da
8.60e31.02
FLI_W03052010_raw004 1: TOF MS ES+ 475.216
8.79e31.02
0.900.860.18 1.191.16 1.921.771.611.30
1.872.07
FLI_W03052010_raw004 1: TOF MS ES+ TIC
3.38e61.19
1.881.85Scan: 200 - 800
MS window: 1 Da
MS window: 0.1 Da
MS window: 0.01Da
MS window: 0.001 Da
58October 14, 2010 GBMSDG Talk
• “Fish-out” drug-derived peaks from endogenous peaks in a complex biological matrix
• Key utility in non-radio-labeled drug-administration
• An alternative to triple quadrupole tools: neutral loss scan (NLS) and precursor ion scan (PIS)
Use of Mass Defect Filter for Post-Acquisition Processing of Accurate Mass (High Resolution) LC-MS Data.
M. Zhu et al., Drug Metab. Dispos. 2006, 34, 1722-1733
59October 14, 2010 GBMSDG Talk
Mass Defect Filter Mass Defect Filter ReferenceReference
J. Mass Spectrom., 2009, 44, 999-1016.
60October 14, 2010 GBMSDG Talk
Exact Mass and Isotopic Abundance of Common Exact Mass and Isotopic Abundance of Common ElementsElements
Element Nuclide Nominal Exact Mass IsotopicMass Mass Defect Abundance
Hydrogen H 1 1.0078 0.00783 100.00%D 2 2.0141 0.0141 0.02%
Carbon C12 12 12.0000 0 100.00%
C13 13 13.0034 0.00336 1.10%
Nitrogen N14 14 14.0031 0.003074 100.00%
N15 15 15.0001 0.0001 0.37%
Oxygen O16 16 15.9949 -0.0051 100.00%
O17 17 16.9991 -0.0009 0.04%
O18 18 17.9992 -0.0008 0.20%
Fluorine F19 19 18.9984 -0.0016 100.00%
Phosphorus P31 31 30.9738 -0.0262 100.00%
Sulfur S32 32 31.9721 -0.0279 100.00%
S33 33 32.9725 -0.0275 0.79%
S34 34 33.9679 -0.0321 4.40%
Chlorine Cl35 35 34.9689 -0.0311 100.00%
Cl37 37 36.9659 -0.0341 32.00%
Bromine Br79 79 78.9183 -0.0817 100.00%
Br81 81 80.9163 -0.0837 97.30%
Iodine I127 127 126.9045 -0.0955 100.00%
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(B)
(C)
Source: JMS 2003, 38, 1110-1112
Mass Defect Filter ExampleMass Defect Filter Example
TIC
14C
Processed MS
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Mass spectra of metabolite x at retention time 35.5 min
(A) Full scan spectrum of metabolite x form the unprocessed total ion chromatogram . (B) Detail of (A) in mass range 450-550 Da.
(C) Full scan spectrum of metabolite x (the molecular ion was at m/z 503.0737 from the MDF processed total ion chromatogram.
(B)
(C)
Source: JMS 2003, 38, 1110-1112
Mass Defect Filter ExampleMass Defect Filter Example
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Metabolite ID Software Metabolite ID Software Tool: Tool:
BgS-NoRABgS-NoRA
Published in RCMS, 23, 1563 (2009).
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Metabolite ID Software Metabolite ID Software Tool: Tool:
BgS-NoRABgS-NoRAMouse urine spiked with diclofenac microsomal incubation sample
TIC
TIC after Background Subtraction
TIC after BgS-NoRA
P = parent
M1, M2, M3 = metabolites
65October 14, 2010 GBMSDG Talk
Metabolite ID Software Metabolite ID Software Tool: Tool:
BgS-NoRABgS-NoRAMouse urine spiked with diclofenac microsomal incubation sample—peak at 7.8 min
Unprocessed data
Mass spectrum after data processed with BgS-NoRA
66October 14, 2010 GBMSDG Talk
Which tool is Best for Which tool is Best for Metabolite ID?Metabolite ID?
Published in RCMS, 24, 939 (2010).
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Additional Development Additional Development StagesStages
that use MS Analysis that use MS Analysis
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TK Study Support TK Study Support Typical study is one compound dosed oral (PO) in Typical study is one compound dosed oral (PO) in
a laboratory animal. The goal is to get TK a laboratory animal. The goal is to get TK (toxicokinetic) parameters. (toxicokinetic) parameters.
This is a GLP (Good Laboratory Practices) study.This is a GLP (Good Laboratory Practices) study. Sample preparation is typically SPE.Sample preparation is typically SPE. A multipoint standard curve is prepared for the A multipoint standard curve is prepared for the
assay.assay. Analysis is by LC-MS/MS on a triple quadrupole Analysis is by LC-MS/MS on a triple quadrupole
MS/MS system. Usually a SIL (stable isotope MS/MS system. Usually a SIL (stable isotope label) internal standard is used for the assay.label) internal standard is used for the assay.
69October 14, 2010 GBMSDG Talk
Clinical PK Study Clinical PK Study SupportSupport
Typical study is one compound dosed oral (PO) Typical study is one compound dosed oral (PO) in humans. The goal is to get PK parameters. in humans. The goal is to get PK parameters.
This is a treated as a GLP (Good Laboratory This is a treated as a GLP (Good Laboratory Practices) study.Practices) study.
Sample preparation is typically SPE.Sample preparation is typically SPE. A multipoint standard curve is prepared for A multipoint standard curve is prepared for
the assay.the assay. Analysis is by LC-MS/MS on a triple Analysis is by LC-MS/MS on a triple
quadrupole MS/MS system. Usually a SIL quadrupole MS/MS system. Usually a SIL (stable isotope label) internal standard is used (stable isotope label) internal standard is used for the assay.for the assay.
70October 14, 2010 GBMSDG Talk
Impurities and Impurities and DegradantsDegradants
These studies are performed to support These studies are performed to support safety studies or clinical studies.safety studies or clinical studies.
The goal is to measure any significant The goal is to measure any significant impurities or degradants that are in the impurities or degradants that are in the pharmaceutical test compound batch pharmaceutical test compound batch used for these studies.used for these studies.
Generally, one would use a combination Generally, one would use a combination of triple quadrupoles as well as QTOF of triple quadrupoles as well as QTOF MS systems as well as the Orbitrap MS MS systems as well as the Orbitrap MS system to characterize these system to characterize these compounds.compounds.
71October 14, 2010 GBMSDG Talk
MS Imaging—a Specialty MS Imaging—a Specialty use of MSuse of MS
0
50
100
150
200
250
300
1970 1980 1990 2000 2010
Num
ber
of p
ublic
ation
s
Year
Increase in imaging MS publications
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MS Imaging using the MALDI QqTOF
QuadrupoleMass Filter
Collision Cell Time-of-
FlightAnalyzer
Detectorh
+
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Optical Image
Radioautographic Image
MALDI-MS/MS Image
1000 µm
•MALDI-MS/MS Image is in good agreement with the radioautographic image
Rat Brain Tissue Slice
--Rat dosed with clozapine
Hsieh Y, et al., Rapid Commun Mass Spectrom. 2006;20(6):965-72.
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Mass Spectral Data Mass Spectral Data Confirms the Presence of Confirms the Presence of
ClozapineClozapine
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Next Step: Whole Mouse SliceNext Step: Whole Mouse Slice MS Imaging MS Imaging
(a) Animal administration (b) Whole-body tissue slicing
(f) MALDI-IMS (e) Sample preparation
(c) Sample transfer to a tape
(d) Adhesive to a MALDI plate
(a) Animal administration (b) Whole-body tissue slicing
(f) MALDI-IMS (e) Sample preparation
(c) Sample transfer to a tape
(d) Adhesive to a MALDI plate
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liver
stomachGI tract
Ion image of fexofenadine (metabolite).
Ion image of terfenadine (parent).
Optical image of whole-body mouseslice
Mouse Whole Body Image—Mouse Dosed with Terfenadine
Result: These MS images allow us to “visualize” first-pass metabolism.
Source: Chen et al.,
Drug Metab. Lett., 2,
1-4 (2008)
100 mpk p.o. 100 mpk p.o. and sacrificed and sacrificed 4 h post4 h post dosingdosing
77
Description of Technology
Automated liquid extraction-based surface sampling technique utilizing the robotic Advion Nanomate chip-based nanoelectrospray platform. Method invented at Oakridge National Laboratory (ORNL). Developed and commercialized at Advion.
Liquid microjunction created between the robotically controlled pipette tip dispensing solvent and surface (e.g. tissue section, blood spots on paper).
The microfluidics chip contains an array of nanoelectrospray nozzles etched in a silicon wafer eliminating carryover as one tip and one nozzle is used per sample.
Can be used for a variety of samples including tissue sections, dried blood spots on paper, samples on MALDI plates for complimentary information by electrospray ionization (ESI), TLC plates, and other planar separation media.
Advion Nanomate LESA (Liquid Extraction Surface Analysis) System
Advion Nanomate ESI Chip
Liquid microjunction between pipette tip and tissue surface
Schematic of chip based nano-ESI infusion http://www.advion.com/biosystems/triversa-nanomate/LESA/index.php
GBMSDG TalkOctober 14, 2010
78
Whole Body Distribution of a Drug and itsMetabolites by QWBA vs LESA
T
7.5 mg/kg IV ‘Cold’ Propranolol
MS Tissue Imaging/Sampling: Sections transferred to glass slides with UV-activated adhesive or collected on adhesive tape and sent to ORNL for MS tissue imaging/profiling
7.5 mg/kg IV [3H] Propranolol
QWBA (Quantitative Whole Body Autoradiography): QWBA study with metabolite ID by radioprofiling in conjunction with nanospray MS or accurate mass MS at Merck Male CD-1 Mice
Male CD-1 Mice
T
T
79
QWBA
Brain Lung Liver Stomach Kidney 20 µM-eq 40 µM-eq 21 µM-eq 31µM-eq 45 µM-eq
Autoradioluminograph [3H]Propranolol Drug Related Material
40 µm Mouse Whole Body Sagittal Section: 60 min post [3H]Propranolol IV Dose
QWBA Results Confirmed High Levels of [3H] Propranolol Related Material in Brain, Lung, Liver, and Kidney
0 100
GBMSDG TalkOctober 14, 2010
80
Identification of [3H] Drug Related Material (DRM) in Tissues
Unchanged parent detected in lung and brain.
Major metabolites in liver and kidney identified as hydroxypropranolol glucuronide metabolites by LC-MS/MS-rad.
GBMSDG TalkOctober 14, 2010
81
Normal Operation using the Advion Nanomate System
Mass spectrometer
Sample
Sampling tip
Nozzle
Aspirate sample
Transfer sample
Apply HV, spray voltage
GBMSDG TalkOctober 14, 2010
82
Operation using the Nanomate Systemfor Surface Sampling – ORNL Invention
(Advion LESA)Mass
spectrometer
SampleOn
Surface
Solvent
Sampling tip
Aspirate solventDispense solvent on
sampleAspirate sample
solutionTransfer sample
Spray sample
Nozzle
Vilmos Kertesz, Gary J. Van Berkel „Fully Automated Liquid Extraction-Based Surface Sampling and Ionization Using a Chip-Based Robotic Nanoelectrospray Platform” J. Mass Spectrom., 2010 Mar;45(3):252-60.
~ 1 mm spot size
4 6 8 10 12 14
Inte
nsity
, cp
s
0
5000
10000
15000
20000
14 16 18 20 22 24
Inte
nsity
, cp
s
0
5000
10000
15000
20000
4 6 8 10 12 14
Inte
nsity
, cp
s
0
5000
10000
15000
20000
14 16 18 20 22 24
Inte
nsity
, cp
s0
5000
10000
15000
20000
LESA: MS for Detection of Propranolol (7.5 mg/kg IV) and a Major Metabolite in Tissues
Dosed tissue Control tissue
stomach
liverlung
kidneymuscle
brain
kidney musclebrain
lungliver
stomach
Propranolol Propranolol
Hydroxypropranolol glucuronide Hydroxypropranolol glucuronide
Lung
Liver Stomach
KidneyBrainMuscle
Lung
LiverKidney
Muscle
t,min t,min
• Rapid and automated technique to sample tissues including ones on tape used for QWBA.
• Successful detection of propranolol and its major glucuronide metabolite not seen by DESI-MS.
StomachBrain
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ConclusionsConclusions
Mass spectrometry is used at multiple Mass spectrometry is used at multiple stages of new drug discovery and stages of new drug discovery and development. Various types of mass development. Various types of mass spectrometers are utilized including spectrometers are utilized including single quadrupoles, triple quadrupoles, single quadrupoles, triple quadrupoles, Q-Traps, Q-TOFs and Orbitrap MS Q-Traps, Q-TOFs and Orbitrap MS systems. The need for the multiple types systems. The need for the multiple types of MS system is due to the variety of of MS system is due to the variety of assays that are mandated at the different assays that are mandated at the different stages in the new drug discovery process.stages in the new drug discovery process.
2008 2009
MS Reference Books
Available at Amazon.com20092005
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Acknowledgments Acknowledgments (Thanks to the following (Thanks to the following for one or more slides)for one or more slides)
Waters-Waters-MicroMassMicroMass
Thermo-FisherThermo-Fisher AB-SciexAB-Sciex AgilentAgilent Marissa VavrekMarissa Vavrek Rick KingRick King
Swapan Swapan ChowdhuryChowdhury
Joanna Zgoda-Joanna Zgoda-PolsPols
Michelle ReyzerMichelle Reyzer Yunsheng HsiehYunsheng Hsieh Fangbiao LiFangbiao Li
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AcknowledgementsAcknowledgements
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Thank you for your attention!Thank you for your attention!
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