1 October 14, 2010 GBMSDG Talk Mass Spectrometry as the Premier Analytical Tool in Drug Discovery and Drug Development Walter Korfmacher Exploratory Drug

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


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

2000000

4000

145

1

0.1

1

10

100

1000

10000

100000

1000000

10000000

100000000

1 2 3 4 5

Approved Drug

Clinical

Development

Compound Libraries

Lead Selection and Optimization

Stages of Discovery and Development

Num

ber

of

com

poun

ds

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


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.


The SolutionThe Solution

LC-MS and LC-MS/MSLC-MS and LC-MS/MS


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


The ChallengeThe Challenge

Choosing the right tool for the task :Choosing the right tool for the task :

MS Toolbox

Hammer?

OR

Wrench?


Common MS Tools


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


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.


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.


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.


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.


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


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)


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!!


RT: 0.00 - 5.02

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

Time (min)

0

10

20

30

40

50

60

70

80

90

100

0

10

20

30

40

50

60

70

80

90

100

Re

lativ

e A

bu

nd

an

ce

2.99

4.213.04

3.913.53

2.853.12

4.36

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


RT: 0.00 - 5.02

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lativ

e A

bu

nd

an

ce

2.83

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


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).


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


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



3A4 2D6 2C9

incubation

detection LC-MS-MS

P450 source human liver microsomes



3A4 2D6 2C9

Method:


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


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


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


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


0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9Time, min

0.0

2.0e4

4.0e4

6.0e4

In

te

ns

it

y,

c

ps

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


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.


CYP Inhibition Screen CYP Inhibition Screen ReviewReview


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.


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


Metabolic Stability Assay Metabolic Stability Assay ExampleExample


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.


Metabolic Stability AssayMetabolic Stability Assay

Scheme shows how a well organized system is needed to provide high throughput metabolic stability data.


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.


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.


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.


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


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


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


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.


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


Typical Discovery PK Typical Discovery PK AssayAssay

1 – 10,000 ng/mL

Res

pons

e ra

tio


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.



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


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


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


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

0 5 10 15 20 25 30 35 40 45

Time (min)

0

20

40

60

80

100

LC/MS Chromatogram

0 5 10 15 20 25 30 35 40 45

Time (min)

0

20

40

60

80

100

Radiocarbon Chromatogram

10-50%

50-90%

Radioactivity helps to locate the metabolites in the samples



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.



(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


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


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


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.


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


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


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

0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00

%

0

100

0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00

%

0

100

0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00

%

0

100

0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00

%

0

100

FLI_W03052010_raw004 1: TOF MS ES+ 475.216 0.00Da

2.90e31.02


2.90e31.02


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


• “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


Mass Defect Filter Mass Defect Filter ReferenceReference

J. Mass Spectrom., 2009, 44, 999-1016.


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%


(B)

(C)

Source: JMS 2003, 38, 1110-1112

Mass Defect Filter ExampleMass Defect Filter Example

TIC

14C

Processed MS


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


Metabolite ID Software Metabolite ID Software Tool: Tool:

BgS-NoRABgS-NoRA

Published in RCMS, 23, 1563 (2009).



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



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


Which tool is Best for Which tool is Best for Metabolite ID?Metabolite ID?

Published in RCMS, 24, 939 (2010).


Additional Development Additional Development StagesStages

that use MS Analysis that use MS Analysis


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.


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.


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.


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


MS Imaging using the MALDI QqTOF

QuadrupoleMass Filter

Collision Cell Time-of-

FlightAnalyzer

Detectorh

+

73October 14, 2010GBMSDG Talk

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.


Mass Spectral Data Mass Spectral Data Confirms the Presence of Confirms the Presence of

ClozapineClozapine


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


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


http://www.advion.com/biosystems/triversa-nanomate/LESA/index.php

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


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.


81

Normal Operation using the Advion Nanomate System

Mass spectrometer

Sample

Sampling tip

Nozzle

Aspirate sample

Transfer sample

Apply HV, spray voltage


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


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


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


AcknowledgementsAcknowledgements


Thank you for your attention!Thank you for your attention!

?

Documents

1 October 14, 2010 GBMSDG Talk Mass Spectrometry as the Premier Analytical Tool in Drug Discovery and Drug Development Walter Korfmacher Exploratory Drug