Transcript GBMSDGKorfmacher1010.pps

Mass Spectrometry as the Premier
Analytical Tool in Drug Discovery
and Drug Development
Walter Korfmacher
Exploratory Drug Metabolism
Merck Research Laboratories
Kenilworth, NJ USA
October 14, 2010
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Outline







New Drug Discovery Challenges
Mass Spectrometry Basics
Selected In vitro Drug Metabolism
Applications
Selected In vivo Drug Metabolism
Applications
Metabolite ID Applications
MS Imaging Applications
Conclusions
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Topics Not Covered




Proteomics
Biomarker discovery or assay
Metabolomics
High Throughput Screening
October 14, 2010
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Drug Discovery:
From Library to Market
Lead
Optimization
Early Discovery
100000000
10000000
Safety Testing
Clinical
Testing
FDA
Approval
Compound Libraries
Lead Selection and Optimization
2000000
1000000
Clinical
100000
4000
Number of
compounds
10000
Development
1000
100
14
10
Approved
Drug
5
1
1
0.1
1
2
3
4
5
Stages of Discovery and Development
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NEW DRUG DISCOVERY PIPELINE
Chemistry
Biology--HTS for Receptor Activity
In-vitro Stability Screen
DMPK
Lead
Optimization
In-vitro Absorption Screen
P450 Enzyme Inhibition Screen
CARRS Oral PK Screen
Rat IV / PO PK
Dog and Monkey IV / PO PK
Metabolite ID
Rising Dose and Multiple Dose
Studies and Safety Screens
Drugs into Development
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The Challenge

How to deal with the multiple compounds at
multiple stages in the drug discovery/drug
development pipeline.
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The Solution

LC-MS and LC-MS/MS
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Why use Mass Spectrometry?
 Specificity!
Non-specific techniques, such as
UV and fluorescence, are unable to provide
proof of the analyte identity
 Ease
of Use! Modern mass spectrometry
software interfaces are easy to use
 Versatility!
MS is both a qualitative and
quantitative technique
October 14, 2010
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The Challenge

Choosing the right tool for the task :
OR
Wrench?
MS Toolbox
Hammer?
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Common MS Tools
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What is LC-MS?
Liquid Chromatography Coupled to a Mass
Spectrometer
(In this case the Mass Spectrometer is a Single Quadrupole
instrument)
HPLC
Column
Ion source
Mass analyzer Detector
APCI or ESI
October 14, 2010
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The Challenge—Compound
Synthesis

At a big Pharma site, one might find hundreds
of medicinal chemists who might produce 2001000 new compounds each week. These have to
be assayed.
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The 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.
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The Challenge—Lead Optimization
In Vitro Screening

At a big Pharma site, one might get 100-200 new
compounds each week that have to be screened
in various in vitro assays. These have to be
assayed separately for each screen.
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The 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.
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What is LC-MS/MS aka Triple
Quadrupole Technology?
Liquid Chromatography Coupled to a Tandem
Mass Spectrometer (In this case the Mass Spectrometer
is a Triple Quadrupole instrument)
Q1
HPLC Column
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Ion source
Q2
Mass analyzer
GBMSDG Talk
Q3
Detector
16
Quantitation Gold Standard:
MS/MS – Selected Reaction Monitoring
(SRM)*
Select
precursor ion in Q1
e.g. m/z 216
Fragment
precursor ion in Q2
(Collision Cell)
Select
product ion in Q3
e.g. m/z 174
* Often referred to as Multiple Reaction Monitoring (MRM)
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Effects of Stages of Analysis on
Signal, Noise, and Signal-to-Noise
Important Concept!!
S/N
Signal
Noise
1
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LC
2
LC-MS
3
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Stages of Analysis
LC-MS/MS
4
?
18
RT: 0.00 - 5.02
90
3.53
70
60
2.85
TIC MS
plasma01
4.21
3.04
LC-TIC
80
Relative Abundance
NL:
1.35E7
2.99
100
3.91
3.12
50
40
4.36
30
2.26 2.63
2.17
20
10
0.22 0.42 0.61
1.01
1.37
1.81
2.00
4.62
0
100
NL:
3.81E5
m/z=
373.0375.0 MS
plasma01
2.83
90
LC-MS
80
70
60
50
40
30
20
2.68
1.90 1.98 2.32 2.56
10
0.32 0.50
0
0.0
0.5
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1.01
1.0
1.30
1.5
2.0
2.5
Time (min)
2.95
3.0
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3.57 3.68 3.75
3.5
4.0
4.38 4.47
4.72
4.5
5.0
19
RT: 0.00 - 5.02
NL:
3.22E3
2.83
100
TIC F: + c APCI
SRM ms2
[email protected] [
120.70-121.30]
MS
npy086G_004
95
90
LC-MS/MS
85
80
75
70
Relative Abundance
65
60
55
50
45
10 ng/ml
40
35
30
25
20
15
10
5
0.42 0.69 1.05
0
0.0
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0.5
1.10
1.0
1.14
1.5
2.04 2.09
2.0
2.74
2.61
2.5
Time (min)
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3.01
3.0
3.60 3.80
3.5
3.89 3.93
4.34 4.56
4.0
4.5
5.0
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Discovery In Vitro Screening




P450 Assay--Enzyme Inhibition Screen
Caco-2 cells—Absorption Screen
Liver Microsomes/Hepatocytes--Metabolic
Stability Screen
Plasma Protein Binding
Each In Vitro Assay Uses LC-MS/MS for the
analytical step (typically a triple quadrupole MS
system).
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High Throughput CYP Inhibition
Assay Example
•
Generic LC-MS/MS method
•
•
1 minute gradient
Monitors 3 substrates in a single LC-MS/MS run
•
Template is used for creating sample list
•
Automatic results calculation and import into
Activity Base
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In Vitro Evaluation of CYP Inhibition
Purpose:
Evaluate direct and mechanism-based inhibitors for P450 enzymes
(3A4, 2D6, 2C9) to assess potential for drug-drug interactions.
Method:
P450 source
human liver microsomes
incubation
cocktail (
substrates
testosterone
3A4, 2D6, 2C9)
Dextromethorphan
3A4
products
detection
October 14, 2010
2D6
6b-hydroxytestosterone
dextrophan
Tolbutamine
2C9
4-hydroxytobutamide
LC-MS-MS
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In Vitro Evaluation of CYP Inhibition
incubation/pre-incubation
Stock
solution
from CDC
Serial dilution
1. Coincunation
2. Coincubation
•three concentrations/cpd: 20 mM, 2 mM and 0.2 mM
3. Preincubation
•duplicates/each conc.
•30 compounds/set
4. Preincubation
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I
n
t
e
LC-MS/MS for p450 Inhibition
Screen-Run time is less than 1 minute
n
XIC of +MRM (4 pairs): 305.2/269.4 amu from Sample 27 (616005) of 090616 3in1 Hui 4 plates.wiff (Heated Nebulizer), Smoothed
I s n
1.29e5
1.00e5
Max. 1.3e5 cps.
0.76
Substrate for CYP3A4, 6b-hydroxytestosterone
i
m/z 305  269
tt
5.00e4
ey
0.00
,n
0.5
0.6
Time, min
XIC of +MRM (4 pairs): 258.1/157.1 amu from Sample 27 (616005) of 090616 3in1 Hui 4 plates.wiff (Heated Nebulizer), Smoothed
I cs n
3.8e5
3.0e5
pi tt
2.0e5
eys
1.0e5
0.0
0.1
0.2
0.3
0.4
,n
I cs n
pi
1.5e5
t
1.0e5
yes
5.0e4
0.0
0.8
0.9
Max. 3.8e5 cps.
0.61
Substrate for CYP2D6, dextrophan
0.1
0.2
0.3
m/z 258  157
0.5
0.6
Time, min
XIC of +MRM (4 pairs): 347.2/121.3 amu from Sample 27 (616005) of 090616 3in1 Hui 4 plates.wiff (Heated Nebulizer), Smoothed
2.0e5
0.7
0.4
0.7
0.8
0.9
Max. 2.0e5 cps.
0.81
Internal Standard
0.1
0.2
m/z 347  121
0.3
0.4
,n
0.5
0.6
Time, min
XIC of +MRM (4 pairs): 287.0/170.9 amu from Sample 27 (616005) of 090616 3in1 Hui 4 plates.wiff (Heated Nebulizer), Smoothed
0.7
0.8
0.9
Max. 7.1e4 cps.
cs
0.69
6.0e4
pi
Substrate for CYP2C9, 4-hydroxytolbutamide
m/z 287  171
4.0e4
t
ys
2.0e4
0.0
0.74
0.1
0.2
0.3
0.4
,
0.5
Time, min
0.6
0.7
0.8
0.9
c
p
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s
CYP Inhibition Screen Throughput
 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.
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CYP Inhibition Screen Review
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The Challenge— Metabolic Stability
Screening


At a big Pharma site, one might get 100-200 new
compounds each week that have to be screened
for metabolic stability.
The challenge is that LC-MS/MS methods
have to be developed for each compound.
October 14, 2010
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The Solution—LC-MS/MS +
Software + 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)
• QuanOptimise™ (Waters-Micromass)
• DiscoveryQuant™ (AB-Sciex)
• OptimizerTM (Agilent)
• Use robots for automated sample handling
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Metabolic Stability Assay Example
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Metabolic 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.
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Metabolic Stability Assay
Scheme shows how a well organized system is needed
to provide high throughput metabolic stability data.
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In Vivo Assays

Various types of in vivo assays are performed as
part of new drug discovery and development

The goal may be to understand absorption (A),
distribution (D), metabolism (M), or excretion
(E) properties of a compound

The goal may be to get pharmacokinetic (PK)
information on a compound
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ADME-PK Studies
D
Brain--
Drug Levels—
Dose NCE
(Drug) PO/IV
Plasma—
A
Drug Levels—
LC-MS/MS
LC-MS/MS
MS Image—
PK Parameters
MALDI-MS/MS
Liver—
D
Drug Levels—
LC-MS/MS
Ref: “Using Mass Spectrometry for Drug Metabolism Studies”
ASMS 2010
W. Korfmacher, ed., CRC
Press, 2005.
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The Challenge— In Vivo PK
Screening


At a big Pharma site, one might get 50 - 100 new
compounds each week that have to be screened
for in vivo PK.
The challenge is that LC-MS/MS methods
have to be developed for each compound.
October 14, 2010
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The Solution—LC-MS/MS +
Software + Planning
• Use a generic HPLC method. This works for 8090% of the compounds.
• Use a vendor-supplied software tool for
automated MS/MS method development.
• Use robots for automated sample handling.
• Develop a standard PK screening assay.
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In Vivo PK Screening
Source: Drug Discovery Today Volume 13, Numbers 7/8 April 2008
Authors: 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
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PK Screening Example: CARRS
• Each Compound  PO Dose + collect 6 time points on two Rats.
Drug Concentration
• Pool the samples across the two rats  6 samples for assay.
The Rapid Rat
RR
0
1
2
3
4
5
6
Hours
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)
Throughput: Up to 96 compounds per week
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CARRS 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)
Rat samples
Standards
25 250 2500
6 Samples
Standards
25 250 2500
Compound 1
2
3
4
6
5
0 standards
Solvent Blanks
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Preclinical PK Studies





Typical study is one compound dosed oral (PO) and IV
(Intravenous) in a laboratory animal. The goal is to get
PK parameters in various preclinical species.
Typically this produces 50-60 plasma samples.
Sample preparation is protein precipitation.
A multipoint standard curve is prepared for the assay
Analysis is by LC-MS/MS on a triple quadrupole
MS/MS system. Usually a generic internal standard is
used for the assay.
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Discovery PK Analysis Flowchart
Discovery PK Analysis Flowchart
Animal Dosing
Protocol
Collect Plasma
Samples in a
96-well plate
Dose Animal
Robotic Transfer of Aliquots to Assay
96-well Plate
96-well plate
containing plasma
standards and samples
The MS/MS
instrument is
normally a triple
quadrupole
system
Weigh Standard and
make stock solution
Manual or Robotic
Transfer of Plasma
standards to Assay
96-well plate
Prepare diluted solutions and
plasma standards using robot
LC-MS/MS system
96-well plate
autosampler
Robotic Sample
Preparation
Set up Method and enter sample list
Send results to database
Prepare PK
report
Distribute the PK
report to the
Discovery Team via
E-mail
Print raw data and assay report
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Rapid MS Method Development ion a Discovery Environment
Samples
SRM
Fail
OK
LC-MS/MS test run (PP Sample prep.)
#1: Matrix effect
Revised Chromatography
#2: Interference
:
LLE
Enhanced mass
resolution
SPE
#3: standard curve linearity
Xu et al. Anal.
Chem.--2005
Assay
October 14, 2010
Non-routine
options
GBMSDG Talk
Response ratio
Typical Discovery PK Assay
1 – 10,000 ng/mL
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Discovery Metabolite ID

Generally this has two components:
Lead Optimization Phase--would use unlabelled
compounds and in vitro samples to look for major
routes of metabolism.
 Pre-Recommendation Phase—Look for problem
metabolites plus in vitro comparison of human to
animal metabolism.

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Discovery Metabolite ID

Mass Spectrometry serves two purposes:


Finding metabolites-MS systems can be used in
various ways to find metabolites in multilple
biological matrices (e.g., plasma, bile, urine).
Structure elucidation—MS systems can be used to
to obtain partial or complete structural
identification for the metabolites
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Triple Quad Scan Functions:
to find metabolites

Neutral Loss Scan


Precursor Ion Scan


no prior knowledge of the parent is required—this is used to
look for certain classes of metabolites (e.g., glucuronide,
sulfate or glutathione conjugates)
only fragmentation pattern of parent is required—may find
unexpected metabolites
SRM/MRM

the fragmentation pattern of parent is used to predict the
fragment ions for likely metabolites—some vendors have
software tools that make it easy to build a scan set
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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
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GBMSDG Talk
• QTrap MS systems are
excellent tools for
quantitative analyses as well
as for metabolite ID
47
applications
QTrap MS Publication
In Vivo PK Samples

Simultaneously quantifying parent drugs and
screening for metabolites in plasma
pharmacokinetic samples using selected reaction
monitoring information-dependent acquisition
on a QTrap instrument:
Li et al. (Covance), RCMS, 1943, 2005.
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Typical Metabolite Profiling Experiments and Instrumentation
Sample from in vivo
or in vitro studies
Mass Spectrometer
10-50%
100
80
60
Injector
40
HPLC
20
0
0
5
10 15 20 25 30 35 40 45
Time (min)
LC/MS Chromatogram
50-90%
Radiometric Flow detector
100
80
Radioactivity helps to locate
the metabolites in the
samples
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40
20
0
0
5
10 15 20 25 30 35 40 45
Time (min)
Radiocarbon Chromatogram
Product Ion Scan
Select Precursor
Ion
Fragmentation
Scan Products
A key technique for obtaining structural information.
Product ion spectrum of a particular compound
m1+
m2+
m2+
m2+
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Discovery 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.
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MS/MS spectrum of M1
51
What is MIST?
 Mass Spectrometry
 Investigators
 Security
 Trust
 MIST
will ensure job security for MS
metabolite ID experts
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What is MIST?
 Metabolites
 In
 Safety
 Testing
 MIST
will ensure job security for MS
metabolite ID experts
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Key MIST Points
1. Human metabolites that can raise a safety concern are those
formed at
 greater than 10 percent
 of parent drug’s systemic exposure
 at steady state.
2. Metabolites identified only in human plasma or
Metabolites present at disproportionately higher levels in humans
than in any of the animal test species should be considered for
safety assessment.

Bottom line: Find human metabolites and then be sure
they are “covered” in the tox species.
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New MS Tool for Finding
Metabolites: HRMS

High Resolution Mass Spectrometry (HRMS)
has become the tool of choice for finding
metabolites in complex biological matrices.
The improved mass resolution can be used to
differentiate metabolites from endogenous
background
 Software tools can use HRMS to find metabolites
 The accurate mass of a detected metabolite can help
to confirm its identity by leading to its empirical
formula

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Two HRMS Systems
LTQ-Orbitrap
The Orbitrap MS provides
mass resolution of 10,000-100,000
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The TOF MS provides
mass resolution of 10,00050,000
56
Why High Mass Resolution?
TOF-MS of Sidenafil Example
Std 1
FLI_W03052010_raw004
MS window: 0.001 Da
%
100
1: TOF MS ES+
475.216 0.00Da
2.90e3
1.02
0
0.20
FLI_W03052010_raw004
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
2.00
1: TOF MS ES+
475.216 0.01Da
2.90e3
1.02
100
%
MS window: 0.01Da
0
0.20
FLI_W03052010_raw004
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
2.00
1: TOF MS ES+
475.216 0.10Da
8.60e3
1.02
100
%
MS window: 0.1 Da
0
0.20
FLI_W03052010_raw004
0.40
0.60
0.80
1.20
1.40
1.60
1.80
2.00
1: TOF MS ES+
475.216
8.79e3
1.02
%
100
0
1.00
0.18
MS window: 1 Da
0.20
FLI_W03052010_raw004
0.86 0.90
0.40
0.60
0.80
1.16 1.19
1.00
1.30
1.20
1.61
1.40
1.60
1.77
1.87
1.92
1.80
2.07
2.00
1: TOF MS ES+
TIC
3.38e6
1.19
100
%
Scan: 200 - 800
1.85 1.88
0
Time
0.20
0.40
October 14, 2010
0.60
0.80
1.00
1.20
GBMSDG Talk
1.40
1.60
1.80
2.00
57
Use of Mass Defect Filter for PostAcquisition Processing of Accurate
Mass (High Resolution) LC-MS Data.
• “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)
M. Zhu et al., Drug Metab. Dispos. 2006, 34, 1722-1733
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Mass Defect Filter Reference
J. Mass Spectrom., 2009, 44, 999-1016.
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Exact Mass and Isotopic Abundance of Common Elements
Element
Nuclide
Nominal
Mass
Mass
Defect
Isotopic
Abundance
H
D
1
2
1.0078
2.0141
0.00783
0.0141
100.00%
0.02%
Carbon
C12
C13
12
13
12.0000
13.0034
0
0.00336
100.00%
1.10%
Nitrogen
N14
N15
14
15
14.0031
15.0001
0.003074
0.0001
100.00%
0.37%
Oxygen
O16
O17
O18
16
17
18
15.9949
16.9991
17.9992
-0.0051
-0.0009
-0.0008
100.00%
0.04%
0.20%
Fluorine
F19
19
18.9984
-0.0016
100.00%
Phosphorus
P 31
31
30.9738
-0.0262
100.00%
Sulfur
S 32
S 33
S 34
32
33
34
31.9721
32.9725
33.9679
-0.0279
-0.0275
-0.0321
100.00%
0.79%
4.40%
Chlorine
Cl35
Cl37
35
37
34.9689
36.9659
-0.0311
-0.0341
100.00%
32.00%
Bromine
Br79
Br81
79
81
78.9183
80.9163
-0.0817
-0.0837
100.00%
97.30%
I127
127
126.9045
-0.0955
100.00%
Hydrogen
Iodine
October 14, 2010
Exact
Mass
GBMSDG Talk
60
Mass Defect Filter Example
TIC
Processed
MS
(B)
(C)
14C
Source: JMS 2003, 38, 1110-1112
October 14, 2010
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Mass Defect Filter Example
(B)
(C)
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.
October 14, 2010
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Source: JMS 2003, 38, 1110-1112
Metabolite ID Software Tool:
BgS-NoRA
Published in RCMS, 23, 1563 (2009).
October 14, 2010
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Metabolite ID Software Tool:
BgS-NoRA
Mouse urine spiked with diclofenac microsomal incubation sample
TIC
TIC after Background
Subtraction
TIC after BgS-NoRA
P = parent
October 14, 2010
GBMSDG Talk
M1, M2, M3 = metabolites64
Metabolite ID Software Tool:
BgS-NoRA
Mouse urine spiked with diclofenac microsomal incubation sample—peak at
7.8 min
Unprocessed data
Mass spectrum after
data processed with
BgS-NoRA
October 14, 2010
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Which tool is Best for Metabolite ID?
Published in RCMS, 24, 939 (2010).
October 14, 2010
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Additional Development Stages
that use MS Analysis
October 14, 2010
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TK Study Support





Typical study is one compound dosed oral (PO) in a
laboratory animal. The goal is to get TK (toxicokinetic)
parameters.
This is a GLP (Good Laboratory Practices) study.
Sample preparation is typically SPE.
A multipoint standard curve is prepared for the assay.
Analysis is by LC-MS/MS on a triple quadrupole
MS/MS system. Usually a SIL (stable isotope label)
internal standard is used for the assay.
October 14, 2010
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Clinical PK Study Support





Typical study is one compound dosed oral (PO) in
humans. The goal is to get PK parameters.
This is a treated as a GLP (Good Laboratory Practices)
study.
Sample preparation is typically SPE.
A multipoint standard curve is prepared for the assay.
Analysis is by LC-MS/MS on a triple quadrupole
MS/MS system. Usually a SIL (stable isotope label)
internal standard is used for the assay.
October 14, 2010
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69
Impurities and Degradants



These studies are performed to support safety
studies or clinical studies.
The goal is to measure any significant impurities
or degradants that are in the pharmaceutical test
compound batch used for these studies.
Generally, one would use a combination of
triple quadrupoles as well as QTOF MS systems
as well as the Orbitrap MS system to
characterize these compounds.
October 14, 2010
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MS Imaging—a Specialty use of MS
Increase in imaging MS publications
300
Number of publications
250
200
150
100
50
0
1970
1980
1990
2000
2010
Year
October 14, 2010
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MS Imaging using the MALDI QqTOF
h

Detector
+
Quadrupole Collision
Mass Filter Cell
October 14, 2010
GBMSDG Talk
Time-ofFlight
Analyzer
72
1000 µm
Rat Brain Tissue Slice
--Rat dosed with clozapine
Optical Image
Radioautographic Image
•MALDI-MS/MS Image is in
good agreement with the
radioautographic image
MALDI-MS/MS Image
October 14, 2010
Hsieh Y, et al., Rapid Commun Mass Spectrom.
2006;20(6):965-72.
73
GBMSDG Talk
Mass Spectral Data Confirms the
Presence of Clozapine
October 14, 2010
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74
Next Step: Whole Mouse Slice
MS Imaging
(a) Animal administration
(f) MALDI-IMS
October 14, 2010
(b) Whole-body tissue slicing
(e) Sample preparation
GBMSDG Talk
(c) Sample transfer to a tape
(d) Adhesive to a MALDI plate
75
Mouse Whole Body
Image—Mouse Dosed
with Terfenadine
Optical image of whole-body mouse
slice
100
mpk p.o.
and sacrificed
4 h post dosing
Source: Chen et al.,
Drug Metab. Lett., 2,
1-4 (2008)
Ion image of terfenadine (parent).
stomach
GI tract
liver
Ion image of fexofenadine (metabolite).
Result: These MS images allow us to “visualize” first-pass metabolism.
October 14, 2010
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Advion Nanomate LESA (Liquid Extraction Surface
Analysis) System
Description of Technology
Advion Nanomate
ESI Chip
Automated liquid extraction-based surface sampling
technique utilizing the robotic Advion Nanomate chipbased 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).
Liquid microjunction between
pipette tip and tissue surface
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.
Schematic of chip based nano-ESI infusion
October 14, 2010
http://www.advion.com/biosystems/triversa-nanomate/LESA/index.php
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Whole Body Distribution of a Drug and its
Metabolites by QWBA vs LESA
7.5 mg/kg IV [3H] Propranolol
T
T
T
Male CD-1 Mice
QWBA (Quantitative Whole
Body Autoradiography):
QWBA study with metabolite ID by
radioprofiling in conjunction with
nanospray MS or accurate mass
MS at Merck
7.5 mg/kg IV ‘Cold’ Propranolol
MS Tissue Imaging/Sampling:
Male CD-1 Mice
Sections transferred to glass slides
with UV-activated adhesive or
collected on adhesive tape and sent
to ORNL for MS tissue
78
imaging/profiling
QWBA Results Confirmed High Levels of [3H] Propranolol
Related Material in Brain, Lung, Liver, and Kidney
40 µm Mouse Whole Body Sagittal Section: 60 min post [3H]Propranolol IV Dose
QWBA
Brain
Lung
20 µM-eq
40 µM-eq
0
Liver
21 µM-eq
Stomach
Kidney
31µM-eq
45 µM-eq
100
Autoradioluminograph [3H]Propranolol Drug Related Material
October 14, 2010
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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 LCMS/MS-rad.
October 14, 2010
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Normal Operation using the Advion
Nanomate System
Mass
spectrometer
Sampling tip
Nozzle
Aspirate sample
Transfer sample
Apply HV, spray voltage
Sample
October 14, 2010
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Operation using the Nanomate System
for Surface Sampling – ORNL Invention
(Advion LESA)
Mass
spectrometer
Sampling tip
Nozzle
~ 1 mm spot size
Sample
On
Surface
Solvent
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.
Aspirate solvent
Dispense solvent on sample
Aspirate sample solution
Transfer sample
Spray sample
82
LESA: MS for Detection of Propranolol (7.5 mg/kg IV)
and a Major Metabolite in Tissues
• 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.
Lung
Lung
Brain
Kidney
Liver Stomach
Brain
Muscle
Dosed tissue
brain
muscle
liver
Intensity, cps
Intensity, cps
lung
5000
0
4
6
8
10
12
20000
15000
10000
5000
brain
kidney muscle
14
14
Intensity, cps
Intensity, cps
15000
10000
5000
0
8
10
12
t,min
lung
stomach
16
18
20
22
24
Hydroxypropranolol glucuronide
20000
6
liver
0
Hydroxypropranolol glucuronide
4
Muscle
Propranolol
stomach
kidney
20000
10000
Kidney
Stomach
Control tissue
Propranolol
15000
Liver
14
20000
15000
10000
5000
0
14
16
18
20
22 t,min 24
Conclusions

Mass spectrometry is used at multiple stages of
new drug discovery and development. Various
types of mass spectrometers are utilized
including single quadrupoles, triple quadrupoles,
Q-Traps, Q-TOFs and Orbitrap MS systems.
The need for the multiple types of MS system is
due to the variety of assays that are mandated at
the different stages in the new drug discovery
process.
October 14, 2010
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84
MS Reference Books
2009
2008
2005
Available at Amazon.com
2009
Acknowledgments (Thanks to the
following for one or more slides)
Waters-MicroMass
 Thermo-Fisher
 AB-Sciex
 Agilent
 Marissa Vavrek
 Rick King

October 14, 2010
Swapan Chowdhury
 Joanna Zgoda-Pols
 Michelle Reyzer
 Yunsheng Hsieh
 Fangbiao Li

GBMSDG Talk
86
Acknowledgements
October 14, 2010
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87
Thank you for your attention!
?
October 14, 2010
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88