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

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Mass spectrometry as the premier analytical tool in drug discovery and drug development

Mass Spectrometry as the Premier Analytical Tool in Drug Discovery and Drug Development

Walter Korfmacher

Exploratory Drug Metabolism

Merck Research Laboratories

Kenilworth, NJ USA

GBMSDG Talk


Outline

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

GBMSDG Talk


Topics not covered

Topics Not Covered

  • Proteomics

  • Biomarker discovery or assay

  • Metabolomics

  • High Throughput Screening

October 14, 2010

GBMSDG Talk


Drug discovery from library to market

Drug Discovery:From Library to Market

Lead Optimization

FDA

Approval

Clinical Testing

Early Discovery

Safety Testing

Compound Libraries

Lead Selection and Optimization

Clinical

Approved Drug

Development

Number of compounds

Stages of Discovery and Development

GBMSDG Talk

October 14, 2010


Mass spectrometry as the premier analytical tool in drug discovery and drug development

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

GBMSDG Talk


The challenge

The Challenge

  • How to deal with the multiple compounds at multiple stages in the drug discovery/drug development pipeline.

GBMSDG Talk


The solution

The Solution

  • LC-MS and LC-MS/MS

GBMSDG Talk


Why use mass spectrometry

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

GBMSDG Talk


The challenge1

The Challenge

  • Choosing the right tool for the task :

OR

Wrench?

MS Toolbox

Hammer?

GBMSDG Talk


Mass spectrometry as the premier analytical tool in drug discovery and drug development

Common MS Tools

GBMSDG Talk


What is lc ms

What is LC-MS?

Liquid Chromatography Coupled to a Mass Spectrometer

(In this case the Mass Spectrometer is a Single Quadrupole instrument)

HPLCColumnIon sourceMass analyzerDetector

APCI or ESI

GBMSDG Talk


The challenge compound synthesis

The Challenge—Compound Synthesis

  • At a big Pharma site, one might find hundreds of medicinal chemists who might produce 200-1000 new compounds each week. These have to be assayed.

GBMSDG Talk


The solution lc ms

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.

GBMSDG Talk


The challenge lead optimization in vitro screening

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.

GBMSDG Talk


The solution lc ms ms

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.

GBMSDG Talk


What is lc ms ms aka triple quadrupole technology

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)

Q1Q2Q3

HPLCColumnIon sourceMass analyzerDetector

GBMSDG Talk


Quantitation gold standard ms ms selected reaction monitoring srm

Quantitation Gold Standard:MS/MS – Selected Reaction Monitoring (SRM)*

SelectFragmentSelect

precursor ion in Q1precursor ion in Q2product ion in Q3

e.g. m/z 216(Collision Cell)e.g. m/z 174

* Often referred to as Multiple Reaction Monitoring (MRM)

GBMSDG Talk


Effects of stages of analysis on signal noise and signal to noise

Effects of Stages of Analysis on Signal, Noise, and Signal-to-Noise

Important Concept!!

S/N

Signal

Noise

1

2

3

4

LC

LC-MS

LC-MS/MS

?

GBMSDG Talk

Stages of Analysis


Mass spectrometry as the premier analytical tool in drug discovery and drug development

LC-TIC

LC-MS

GBMSDG Talk


Mass spectrometry as the premier analytical tool in drug discovery and drug development

LC-MS/MS

10 ng/ml

GBMSDG Talk


Discovery in vitro screening

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).

GBMSDG Talk


High throughput cyp inhibition assay example

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

GBMSDG Talk


Mass spectrometry as the premier analytical tool in drug discovery and drug development

Method:

Method:

P450 source

P450 source

P450 source

human liver microsomes

human liver

human liver

incubation

incubation

incubation

cocktail (

cocktail

3A4, 2D6, 2C9)

substrates

substrates

substrates

testosterone

testosterone

testosterone

Dextromethorphan

Dextromethorphan

Dextromethorphan

Tolbutamine

Tolbutamine

Tolbutamine

3A4

3A4

3A4

2C9

2C9

2C9

2D6

2D6

2D6

b

b

b

products

products

products

6

6

6

-

-

-

hydroxytestosterone

hydroxytestosterone

hydroxytestosterone

dextrophan

dextrophan

dextrophan

4

4

4

-

-

-

hydroxytobutamide

hydroxytobutamide

hydroxytobutamide

detection

detection

detection

LC

LC

LC

-

-

-

MS

MS

MS

-

-

-

MS

MS

MS

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.

GBMSDG Talk


In vitro evaluation of cyp inhibition

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

  • duplicates/each conc.

  • 30 compounds/set

3. Preincubation

4. Preincubation

GBMSDG Talk


Lc ms ms for p450 inhibition screen run time is less than 1 minute

LC-MS/MS for p450 Inhibition Screen-Run time is less than 1 minute

Substrate for CYP3A4, 6-hydroxytestosterone

m/z 305  269

Substrate for CYP2D6, dextrophan

m/z 258  157

Internal Standard

m/z 347  121

Substrate for CYP2C9, 4-hydroxytolbutamide

m/z 287  171

GBMSDG Talk


Cyp inhibition screen throughput

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.

GBMSDG Talk


Cyp inhibition screen review

CYP Inhibition Screen Review

GBMSDG Talk


The challenge metabolic stability screening

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.

GBMSDG Talk


The solution lc ms ms software hardware

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

  • GBMSDG Talk


    Metabolic stability assay example

    Metabolic Stability Assay Example

    GBMSDG Talk


    Metabolic stability assay

    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.

    GBMSDG Talk


    Metabolic stability assay1

    Metabolic Stability Assay

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

    GBMSDG Talk


    In vivo assays

    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

    GBMSDG Talk


    Adme pk studies

    ADME-PK Studies

    Brain-- D

    Drug Levels—

    LC-MS/MS

    MS Image—

    MALDI-MS/MS

    Plasma—A

    Drug Levels—

    LC-MS/MS

    PK Parameters

    Dose NCE (Drug) PO/IV

    Liver—D

    Drug Levels—

    LC-MS/MS

    Ref: “Using Mass Spectrometry for Drug Metabolism Studies”

    W. Korfmacher, ed., CRC Press, 2005.

    ASMS 2010


    The challenge in vivo pk screening

    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.

    GBMSDG Talk


    The solution lc ms ms software planning

    The Solution—LC-MS/MS + Software + 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 handling.

    • Develop a standard PK screening assay.

    GBMSDG Talk


    In vivo pk screening

    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

    GBMSDG Talk


    Pk screening example carrs

    PK Screening Example: CARRS

    • 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

    GBMSDG Talk


    Carrs assay

    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)

    GBMSDG Talk


    Preclinical pk studies

    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.

    GBMSDG Talk


    Discovery pk analysis flowchart

    Discovery PK Analysis Flowchart

    The MS/MS instrument is normally a triple quadrupole system

    GBMSDG Talk


    Mass spectrometry as the premier analytical tool in drug discovery and drug development

    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

    Non-routine options

    Assay

    Rapid MS Method Development ion a Discovery Environment

    Xu et al. Anal. Chem.--2005

    GBMSDG Talk

    October 14, 2010


    Typical discovery pk assay

    Typical Discovery PK Assay

    Response ratio

    1 – 10,000 ng/mL

    GBMSDG Talk


    Discovery metabolite id

    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.

    GBMSDG Talk


    Discovery metabolite id1

    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

    GBMSDG Talk


    Triple quad scan functions to find metabolites

    Triple Quad Scan Functions:to find metabolites

    • Neutral Loss 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)

    • Precursor Ion Scan

      • 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

    GBMSDG Talk


    Mass spectrometry as the premier analytical tool in drug discovery and drug development

    • 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

    GBMSDG Talk


    Qtrap ms publication in vivo pk samples

    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.

    GBMSDG Talk


    Mass spectrometry as the premier analytical tool in drug discovery and drug development

    Mass Spectrometer

    Sample from in vivo

    or in vitro studies

    10-50%

    Injector

    HPLC

    50-90%

    Radiometric Flow detector

    100

    100

    80

    80

    60

    60

    40

    40

    20

    20

    0

    0

    0

    0

    5

    5

    10

    10

    15

    15

    20

    20

    25

    25

    30

    30

    35

    35

    40

    40

    45

    45

    Time (min)

    Time (min)

    Radiocarbon Chromatogram

    LC/MS Chromatogram

    Typical Metabolite Profiling Experiments and Instrumentation

    Radioactivity helps to locate the metabolites in the samples

    October 14, 2010

    GBMSDG Talk


    Product ion scan

    Product ion spectrum of a particular compound

    m1+

    m2+

    m2+

    m2+

    Product Ion Scan

    Select Precursor

    Ion

    Scan Products

    Fragmentation

    A key technique for obtaining structural information.

    GBMSDG Talk


    Discovery metabolite id2

    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.

    GBMSDG Talk

    MS/MS spectrum of M1


    What is mist

    What is MIST?

    • Mass Spectrometry

    • Investigators

    • Security

    • Trust

    • MIST will ensure job security for MS metabolite ID experts

    GBMSDG Talk


    What is mist1

    What is MIST?

    • Metabolites

    • In

    • Safety

    • Testing

    • MIST will ensure job security for MS metabolite ID experts

    GBMSDG Talk


    Key mist points

    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.

  • GBMSDG Talk


    New ms tool for finding metabolites hrms

    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

    GBMSDG Talk


    Two hrms systems

    Two HRMS Systems

    LTQ-Orbitrap

    The Orbitrap MS provides

    mass resolution of 10,000-100,000

    The TOF MS provides mass resolution of 10,000-50,000

    GBMSDG Talk


    Why high mass resolution tof ms of sidenafil example

    Why High Mass Resolution? TOF-MS of Sidenafil Example

    MS window: 0.001 Da

    MS window: 0.01Da

    MS window: 0.1 Da

    MS window: 1 Da

    Scan: 200 - 800

    GBMSDG Talk


    Mass spectrometry as the premier analytical tool in drug discovery and drug development

    Use of Mass Defect Filter for Post-Acquisition 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

    GBMSDG Talk


    Mass defect filter reference

    Mass Defect Filter Reference

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

    GBMSDG Talk


    Mass spectrometry as the premier analytical tool in drug discovery and drug development

    Exact Mass and Isotopic Abundance of Common Elements

    GBMSDG Talk


    Mass spectrometry as the premier analytical tool in drug discovery and drug development

    Mass Defect Filter Example

    (B)

    TIC

    (C)

    Processed MS

    14C

    Source: JMS 2003, 38, 1110-1112

    GBMSDG Talk


    Mass spectrometry as the premier analytical tool in drug discovery and drug development

    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.

    GBMSDG Talk

    Source: JMS 2003, 38, 1110-1112


    Metabolite id software tool bgs nora

    Metabolite ID Software Tool: BgS-NoRA

    Published in RCMS, 23, 1563 (2009).

    GBMSDG Talk


    Metabolite id software tool bgs nora1

    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

    M1, M2, M3 = metabolites

    GBMSDG Talk


    Metabolite id software tool bgs nora2

    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

    GBMSDG Talk


    Which tool is best for metabolite id

    Which tool is Best for Metabolite ID?

    Published in RCMS, 24, 939 (2010).

    GBMSDG Talk


    Additional development stages that use ms analysis

    Additional Development Stagesthat use MS Analysis

    GBMSDG Talk


    Tk study support

    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.

    GBMSDG Talk


    Clinical pk study support

    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.

    GBMSDG Talk


    Impurities and degradants

    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.

    GBMSDG Talk


    Ms imaging a specialty use of ms

    MS Imaging—a Specialty use of MS

    GBMSDG Talk


    Mass spectrometry as the premier analytical tool in drug discovery and drug development

    Detector

    Quadrupole

    Mass Filter

    Collision

    Cell

    Time-of-

    Flight

    Analyzer

    h

    +

    MS Imaging using the MALDI QqTOF

    GBMSDG Talk


    Mass spectrometry as the premier analytical tool in drug discovery and drug development

    Rat Brain Tissue Slice

    --Rat dosed with clozapine

    Radioautographic Image

    MALDI-MS/MS Image

    1000 µm

    Optical Image

    • MALDI-MS/MS Image is in good agreement with the radioautographic image

    Hsieh Y, et al., Rapid Commun Mass Spectrom. 2006;20(6):965-72.

    GBMSDG Talk


    Mass spectral data confirms the presence of clozapine

    Mass Spectral Data Confirms the Presence of Clozapine

    GBMSDG Talk


    Next step whole mouse slice ms imaging

    Next Step: Whole Mouse Slice MS Imaging

    GBMSDG Talk


    Mass spectrometry as the premier analytical tool in drug discovery and drug development

    Mouse Whole Body Image—Mouse Dosed with Terfenadine

    Optical image of whole-body mouse

    slice

    • 100 mpk p.o.

    • and sacrificed

    • 4 h postdosing

    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.

    GBMSDG Talk


    Advion nanomate lesa liquid extraction surface analysis system

    Advion Nanomate LESA (Liquid Extraction Surface Analysis)System

    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

    ESI Chip

    Liquid microjunction between pipette tip and tissue surface

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

    Schematic of chip based nano-ESI infusion

    October 14, 2010

    GBMSDG Talk


    Mass spectrometry as the premier analytical tool in drug discovery and drug development

    Whole Body Distribution of a Drug and itsMetabolites by QWBA vs LESA

    7.5 mg/kg IV [3H] Propranolol

    T

    QWBA (Quantitative Whole Body Autoradiography):QWBA study with metabolite ID by radioprofiling in conjunction with nanospray MS or accurate mass MS at Merck

    T

    T

    Male CD-1 Mice

    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

    Male CD-1 Mice


    Mass spectrometry as the premier analytical tool in drug discovery and drug development

    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 Liver Stomach Kidney20 µM-eq 40 µM-eq 21 µM-eq 31µM-eq 45 µM-eq

    0 100

    Autoradioluminograph [3H]Propranolol Drug Related Material

    October 14, 2010

    GBMSDG Talk


    Identification of 3 h drug related material drm in tissues

    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.

    October 14, 2010

    GBMSDG Talk


    Normal operation using the advion nanomate system

    NormalOperationusing the Advion Nanomate System

    Mass spectrometer

    Sampling tip

    Nozzle

    Aspirate sample

    Transfer sample

    Apply HV, spray voltage

    Sample

    October 14, 2010

    GBMSDG Talk


    Operation using the nano m ate system for surface sampling ornl invention advion lesa

    Operation using the Nanomate Systemfor Surface Sampling – ORNL Invention (Advion LESA)

    Mass spectrometer

    Sampling tip

    Nozzle

    ~ 1 mm spot size

    Aspirate solventDispense solvent on sampleAspirate sample solutionTransfer sampleSpray sample

    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.


    Mass spectrometry as the premier analytical tool in drug discovery and drug development

    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

    Kidney

    Brain

    Brain

    Liver

    Kidney

    Stomach

    Muscle

    Muscle

    Liver

    Stomach

    Dosed tissue

    Control tissue

    Propranolol

    Propranolol

    stomach

    brain

    kidney

    lung

    muscle

    liver

    brain

    liver

    kidney

    lung

    stomach

    muscle

    Hydroxypropranolol glucuronide

    Hydroxypropranolol glucuronide

    t,min

    t,min


    Conclusions

    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.

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    Mass spectrometry as the premier analytical tool in drug discovery and drug development

    MS Reference Books

    2009

    2008

    2009

    2005

    Available at Amazon.com


    Acknowledgments thanks to the following for one or more slides

    Waters-MicroMass

    Thermo-Fisher

    AB-Sciex

    Agilent

    Marissa Vavrek

    Rick King

    Swapan Chowdhury

    Joanna Zgoda-Pols

    Michelle Reyzer

    Yunsheng Hsieh

    Fangbiao Li

    Acknowledgments (Thanks to the following for one or more slides)

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    Acknowledgements

    Acknowledgements

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    Thank you for your attention

    Thank you for your attention!

    ?

    ?

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