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Division of Chemistry. Robert J. Turesky, Ph.D. Division Director. Ruth York. Dwight Miller, Ph.D. Paul Siitonen. Jack Lay, Jr., Ph.D. Analytical Chemical & Biomarkers. NTP Coordinator. Mass Spectrometry. Shannon Snellings, Ph.D. Lee Holder. Larry Rushing *. J. Pat Freeman, Ph.D.

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Division of chemistry

Division of Chemistry

Robert J. Turesky, Ph.D.

Division Director

Ruth York

Dwight Miller, Ph.D.

Paul Siitonen

Jack Lay, Jr., Ph.D.

Analytical Chemical & Biomarkers

NTP Coordinator

Mass Spectrometry

Shannon Snellings, Ph.D.

Lee Holder

Larry Rushing *

J. Pat Freeman, Ph.D.

Catharina Ang, Ph.D.

Julian Leakey, Ph.D.

Rick Beger, Ph.D.

Tom Schmitt *

Jon G. Wilkes, Ph.D.

F. Evans Ph.D.

Yanyan Cui, Ph.D.

Robert Cecotti, Ph.D.

Ronald Evans

Dan Buzatu, Ph.D.

Wenhong Luo, Ph.D.

Willie Cooper

Rick Holland

Alex Shvartsburg, Ph.D.

Theresa Gehring *

Thomas Heinze

Kenneth Roberts, Ph.D.

Mark Billedeau

Danny Nestorick *

To be filled

To be filled

Eugene Hansen

To be filled

*Multiple activities


Division of chemistry mission statement

Division of Chemistry Mission Statement

To utilize chemical research techniques including analytical chemistry, mass and NMR spectrometry, spectroscopic and computational methods to implement intradivisional, intercenter and FDA relevant research initiatives in toxicology, risk assessment, and regulatory compliance


Key research projects

Key Research Projects

R. Beger, E07068, Spectrometric Data Activity Relationship (SDAR) Models for Compounds Binding to Receptors of Toxic Responses: Predictive Toxicology

F. Evans, E07078, NMR spectroscopy of drug purity and public health implications

J. Lay, E07005, Rapid identification of intact whole bacteria based upon spectral patterns using MALDI-TOF MS

D. Miller, E06874, Fresh Tag SensorTM technology for product safety, quality, and rapid screening of explosives

J. Wilkes, E06931, Rapid screening and identification of complex mixtures by pyrolysis-mass spectrometry with pattern recognition


Key research projects cont

Key Research Projects (cont.)

C. Ang, E07056, Chemical characterization of selected medicinal botanical products

J. Leakey and C. Ang, X00031, Impact of dietary supplements on woman’s health issues

D. Buzatu E07077, Comparison of principal components analysis (PCA) and artificial neural networks (ANN) for the prediction of qualitative and quantitative biological end points from spectrometric data

R. Turesky X……., Risk assessment of dietary contaminants (heteroyclic aromatic amines and mycotoxins)


Division of chemistry

National Toxicology Program Activities

Summary

Reports

Dosage

Form

NTP Study

Dosage Verification

Method Development

Dose Certification

Stability

Homogeneity


Division of chemistry

Surveillance Activities

Rodent

Diets

Drinking

Water

Analyses

Bedding


Division of chemistry

Active Collaborations with FDA

Center for Veterinary Medicine

  • Amoxicillin

  • Erythromycin

  • Lincomycin

  • Sulfa Drugs

All Projects Requiring Development of Determinative Methods that Achieve CVM Method Trial Ruggedness Testing Requirements for Reliability


Division of chemistry

Characterization of Bacteria by MALDI TOF/MS

According to the CDC in 1999, as a direct result of microbial

contamination of food there were:

·

76,000,000 food-borne illnesses in the United States

·

325,000 reported hospitalizations and

·

5,000 deaths

64% of the deaths were from unknown organisms.


Division of chemistry

For V. p. MALDI gives signals that correlate well

with regional outbreaks of seafood pathogens

Strains from the Pacific Northwest are reproducible (see below) but

significantly different from strains associated with the Gulf coast (over).

V. parahaemolyticus -Washington State

Vibrio Parahaemolyticus 10293

9477

p18-a8-vp10293 20 (1.889) Sb (49,1.00 ); Sm (SG, 1x10.00); Cm (20:21)

100

%

9087

9419

8910

0

m/z

8800

8900

9000

9100

9200

9300

9400

9500

9600

9700

9800

9900

10000

9478

Vibrio Parahaemolyticus 10290

p18-a5-vp10290 20 (1.895) Sb (49,1.00 ); Sm (SG, 1x10.00); Cm (20:28)

100

9088

9458

%

8911

9419

0

m/z

8800

8900

9000

9100

9200

9300

9400

9500

9600

9700

9800

9900

10000

m/z


Division of chemistry

The Gulf coast strains have a different spectrum

in this mass region giving a marker ion near 9588. [The similar

mass value does not mean the proteins are related!]

9478.8

Vibrio Parahaemolyticus 10290

100

9088.4

V. parahaemolyticus -Washington State

9458.0

%

8911.1

9419.2

9947.0

8766.3

0

m/z

8800

8900

9000

9100

9200

9300

9400

9500

9600

9700

9800

9900

10000

9587.5

Vibrio Parahaemolyticus 2030

Texas Outbreak

9090.5

100

9459.4

9422.0

%

8912.5

0

8800

8900

9000

9100

9200

9300

9400

9500

9600

9700

9800

9900

10000

m/z


Division of chemistry

Proteomics and Mass Spectrometry

Acid resistance and protein biomarkers in bacteria can be monitored by MALDI TOF MS of intact cells. The ions below from are marker proteins (HdeA and HdeB) from the acid resistance gene.

9060

9735

9060

9735

S. flexneri

E. coli


Division of chemistry

MILESTONES

  • MALDI can differentiate bacteria by genus, species, and strain:

    • J.O. Lay, Jr., “MALDI TOF Mass Spectrometry and Bacterial Taxonomy” Trends in Analytical Chemistry, 19, 507 (2000).

  • Specific Biomarkers for virulence can be detected by MALDI:

    • R.D. Holland, C.R. Duffy, F. Rafii, J.B. Sutherland, T.M. Heinze, C.L. Holder, K.J. Voorhees and J.O. Lay, Jr., “Identification of Bacterial Proteins Observed in MALDI TOF Mass Spectra from Whole Cells”, Anal. Chem.71:3226-3230 (1999).

  • Biomarker proteins can sometimes be detected in contaminated media without pre-MS culture steps:

  • R.D. Holland, F. Rafii, T.M. Heinze, J.B. Sutherland, K.J. Voorhees and J.O. Lay, Jr. “MALDI TOF/MS detection of bacterial biomarker proteins isolated from contaminated water, lettuce and cotton cloth” Rapid Communications in Mass Spectrometry, 14:911 (2000).


  • Division of chemistry

    • Future Experiments:

    • Correlation of toxicity and strain types with MALDI spectra

    • Development of more powerful MS methods (MALDI/FTMS)

    • More accurate assignment of biomarker (protein) identity.

    • Benefits to FDA include:

    • Differentiation of strains from more difficult Vibrio species

    • Detection of biomarkers associated with antibiotic resistance

    • Applications to FDA programs in bioterrorism, proteomics

    • and even characterization of other cell types, possibly malignant cells, by MS.


    Division of chemistry

    Metastable Atom Bombardment Time of Flight Mass Spectrometry (MAB/TOF/MS) an Alternative Approach to Bacterial I.D.

    GOALS and OBJECTIVES

    • Rapid chemotaxonomic strain-specific bacterial identification

    • Development of bacterial databases and search strategies

    • Applications to food/seafood borne bacteria, especially Vibrio species (CFSAN & ORA)

    • Development of patents for new methods

    • Identification of bacteria without a prior cell-culture step


    Key findings to date

    KEY FINDINGS TO DATE

    • Demonstrated that a multiplicity of laboratory variables distort mass spectral fingerprints.

    • Patented a simple algorithm to correct for such method-related spectral changes.

    • The correction is more practical than using identical conditions.

    • (US Pat. App. No. 60/239,549 filed 10/10/2000)


    Division of chemistry

    100

    %

    0

    m/z

    100

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    360

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    460

    480

    500

    Matching a Reference Spectrum and an Experimental Spectrum from a Field Test Using MAB TOF MS

    100

    114

    Bacillus globigii(bioagent simulant) reference spectrum

    162

    98

    %

    272

    168

    136

    124

    254

    186

    192

    204

    218

    284

    255

    242

    236

    362

    341

    386

    410

    0

    ARCA3_15 63 (0.105) Cm (40:121)

    114

    A spectrum of airborne particulate collected down-wind from an actual release of Bacillus globigii by the Canadian Military

    (rapid analysis and no culture step!)

    162

    272

    168

    254

    126

    146

    284

    218

    182

    136

    191

    223

    204

    362

    316

    m/z


    Future directions

    FUTURE DIRECTIONS

    • Experimental

    • -test Py-MAB-TOF- MS (from Dephy, Montreal) at NCTR. For applications in rapid speciations

    • Assemble and validate a 200-sample spectral database using bacteria from CFSAN and ORA reference collections.

    • Computational

    • License the patent on using a spectral correction method to mitigate laboratory-based variations

    • Develop similar algorithms to transform spectra from environmental samples to their equivalent laboratory (data base) spectra


    Division of chemistry

    Exp.# E7080

    "Fresh Tag"

    Fulton Fish Market

    Consumer version

    printable

    In the bag test

    Commercial version

    before

    after


    Division of chemistry

    Exp.# E7080

    Indole test

    Grind 20 g shrimp in 50 mL toluene and 5 mL 5% TCA for 1 minute

    Centrifuge puree for 30 minutes at 3500 rpm and decant off toluene layer

    Filter extract through a 0.45 mm syringe filter into a beaker containing anhydrous Na2SO4

    GC-MS Method

    Colorimetric Method

    shrimp

    Std.


    Division of chemistry

    Exp.# E7080

    Aldehydes & Sulfides test

    GAS PHASE TEST

    Solid phase purpal test

    (DEVELOPMENTAL)

    cod


    Division of chemistry

    Fresh Tag onpaper

    Exp.# E7081

    post exposure

    Pre-exposure

    Component of Explosives

    Ammonium Nitrate

    20 Hr. purge 40 mL/min

    AM

    UNK

    5


    Division of chemistry

    In Collaboration with CFSANMethods Development for Bioactive Herbal Ingredients in Functional Foods

    Research Progress:

    Extraction and LC methods developed for 4 SJW components in tea powder, fortified drinks, puffs and snack bars

    Methods Developed for 5 phenolic compounds in echinacea capsules and tablets


    Potential toxicity of herbal constituents

    Potential Toxicity of Herbal Constituents

    Investigators: J. Leakey, C.Ang, R. Cecotti, Y. Cui.

    Objectives:

    1.To develop human cell-based assays to determine whether a test substance affects key enzymes involved in the metabolism of pharmaceuticals.

    2.To use these assay systems to investigate potential drug-herb interactions between prescribed pharmaceuticals and dietary supplements.


    Preliminary findings

    PRELIMINARY FINDINGS

    • Developed methods for isolating hyperforin, the major active ingredient of St. John’s Wort.

    • Developed or procured battery of cell lines expressing major isoforms of human drug metabolizing enzymes: used in inhibition assays.

    • Established that constituents of Echinacea inhibit enzymes conjugating estrogens.


    Future work

    Future Work

    • Develop human hepatocyte-based assay systems for measuring drug metabolizing enzyme induction.

    • Investigate the metabolism of active ingredients of St. John’s Wort by human enzymes.

    • Apply inhibition and induction assays to other herbal products.

    • Apply gene array technology and ultimately proteomics to elucidate mechanisms of action.

    • Isolate and identify the inhibitory constituents of Echinacea and St. John’s Wort.


    Division of chemistry

    O

    H

    C

    H

    3

    H

    H

    H

    200 150 100 50 0

    H

    O

    Protocol E0706801: Relationship between Structure-Activity Relationship (SAR) and Spectrometric Data-Activity Relationship (SDAR) Modeling

    Spectra

    Structure

    SDAR/QSDAR

    SAR/QSAR

    Biological Activity


    Success of sdar and qsdar models

    SDAR model of 108 compounds binding to the estrogen receptor using NMR and MS data.

    QSDAR model of 26 poly- chlorinated dibenzofurans binding to the aryl receptor using predicted NMR data.

    -3.5

    0.98 -0.50

    Component 2

    Predicted Log EC50

    -9.5

    -0.48 1.1

    Log EC50

    -9.5

    -3.5

    Component 1

    Success of SDAR and QSDAR Models


    Sdar publications and patents

    SDAR Publications and Patents

    • 13C NMR and EI Mass Spectrometric Data to Produce a Predictive Model of Estrogen Receptor Binding Toxicology and Applied Pharmacology. 169: 17-25, 2000.

    • Producing 13C NMR, Infrared Absorption and EI Mass Spectrometric Data Monodechlorination Models of Chlorobenzenes, Chlorophenols, and Chloroanilines J. Chem. Inf. Comput. Sci. 40:1449-1455, 2000.

    • Developing 13C NMR Quantitative Spectrometric Data-activity Relationship (QSDAR) Models to the Corticosteroid Binding Globulin. J. Comput.-Aided Molec. Design.

    • Models of Polychlorinated Dibenzodioxins, Dibenzofurans, and Biphenyls Binding Affinity to the Aryl Hydrocarbon Receptor Developed Using 13C NMR Data. J. Chem. Inf. Comput. Sci.

    • Patent Pending for “Methods for Predicting the Biological, Chemical, and Physical Properties of Molecules From Their Spectral Properties.”


    Future directions of sdar

    Future Directions of SDAR

    • Protocol E0706801: “Continuing to develop SDAR models for the Ames test, neuraltoxicity (Neurotox), and other toxic endpoints”

    • Protocol E0706811: “Developing new strategies for spectrometric models of toxicity” (ROW)

    • Protocol E0708301: “Computational predictive system for rodent organ-specific carcinogenicity” (Biometry, CDER, ROW)

    • Producing hybrid spectrometric models that incorporate three-dimensional structural information directly into the SDAR model.


    Division of chemistry

    O

    N

    N

    H

    O

    H

    H

    N

    HO

    N

    H

    C

    C

    H

    N

    H

    N

    N

    3

    2

    N

    H

    O

    H

    N

    C

    O

    O

    H

    N

    O

    H

    C

    O

    H

    O

    3

    N

    O

    H

    N

    H

    N

    N

    H

    C

    C

    H

    3

    3

    O

    N

    T

    NHSO

    -

    3

    N

    N

    H

    C

    N

    CH

    N

    H

    3

    3

    2

    N

    N

    N

    N

    H

    C

    C

    H

    3

    3

    N

    Risk Assessment, Interspecies Extrapolaton and Predictive Toxicology with Biomarkers and Computational Chemisry

    ?

    ?

    Interspecies extrapolation

    DNA

    A

    T

    A

    T

    G

    C

    C

    G

    DNA adduct

    QSAR

    SDAR

    QSDAR

    in vitro

    Metabolites

    Structure & activity

    Spectra & activity

    Computational

    Chemistry


    Division of chemistry

    Protocol E07077.01: Comparison of Principal

    Components Analysis (PCA) and Artificial Neural

    Networks (ANN) for the Prediction of Qualitative

    and Quantitative Biological End Points from

    Spectrometric Data

    Chemical

    Spectrum

    Artificial Neural Network

    Predicted

    Biological

    End Point


    Division of chemistry

    Success of Quantitative Spectral Data Activity Relationship Artificial Neural Network Model (QSDAR-ANN)

    QSDAR-ANN

    model results of 28

    Poly-chlorinated

    Biphenyl, Dioxin,

    and Furan Toxic

    Equivalence Factors

    (TEFs) using

    predicted 13C NMR

    spectra.


    Division of chemistry

    Publications :

    Predicting Toxic Equivalent Factors from NMR Spectra for Dioxins Furans

    and PCBs Using Principle Components Analysis and Artificial Neural

    Networks, Environmental Health Perspectives, manuscript in

    preparation (2001).

    Future Directions:

    • Currently developing a quantum mechanical parameter based neural network model for the prediction of TEFs for the dioxins and dioxin-like compounds.

    • Development of an internet parallel distributed neural network to allow for the handling of large data sets as well as increasing the efficiency of the neural network.


    Division of chemistry

    A New Approach to the NMR Spectroscopy of Drug

    Purity and the Public Health Implications (E070781)

    Objectives:

    ·

    Determine properties and develop procedures for use

    of NMR spectrometer at the NCTR under high

    dynamic range conditions.

    ·

    Develop concepts and methodology for application of

    NMR spectroscopy to investigation of very-low-level

    impurities in drugs using results on genistein as a model


    Division of chemistry

    MS Instrumentation Available (or Planned) for Proteomics

    InstrumentApplication

    LC Triple Quadrupole /MS ESI MW determination for isolated proteins

    confirmation of MW for peptides/small proteins

    Quadrupole TOF MSMALDI and LC/ESI for sequencing

    especially for tagged proteins in measurement

    or relative levels of expression

    SELDIMALDI of affinity surfaces

    rapid screening of dirty samples for end-point

    specific proteins

    {Offsite}

    MALDI TOF MS {at UAF}MW determination for proteins and digests

    MALDI FTMF {at UAF}more accurate mass assignments and

    analysis of whole cells


    Mass spectrometry applications in fda research initiatives

    Mass Spectrometry Applications in FDA Research Initiatives

    Allergenicity

    Bacteria Taxonomy/Speciation

    Bioterrorism

    Drug Purity (Chemicals and Recombinant Proteins)

    Ion Mobility MS (Protein conformation, configuration)

    Microbial metabolism (biotransformation of drugs, contaminants, and antibiotics/resistance)

    Proteomics

    Quality Assurance and Compliance

    Rapid through-put Analysis

    Redox Status (Vitamins, Lipids, Proteins, DNA)

    Risk Assessment (Biomarkers, DNA- and Protein Adducts, DNA Damage, Metabolites)


    Division of chemistry

    Development of a New Tandem Instrumental Approach to the Detection of Prions: HPLC/IMS/MS

    Chromatographic

    HPLC Mixture Resolution

    (liquid-phase) (protein)

    prion level)

    time(min)

    Mobility Based

    IMS Prion Separation

    (gas-phase) (folding) changes) time (ms)

    MS Based

    MS Prion Detection

    (high-vacuum) m/z (mass confirmation)


    Nmr spectroscopy applications in fda research initiatives

    NMR Spectroscopy Applications in FDA Research Initiatives

    Computational Chemistry

    Metabolomics

    Drug Purity

    Proteomics

    LC-NMR-MS


    Division of chemistry

    In vivo NMR for Detection of Biomarkers and the Intermediates of Metabolic Pathways

    Example: Downs Syndrome

    13CbH2-

    Serine

    515N-THF

    Met

    5,10-13CH2-THF

    HCN NMR experiment can monitor 5,10-13CH2-THF and515N-13CH3-THF compounds

    Homo-

    Cysteine

    515N-13CH3-

    THF

    • Cost to Upgrade NMR ~ $350,000 or New NMR ~ $550,000

    • Cost of labeled compounds ~ $15,000/year


    Areas supported in fy 2001

    Areas Supported in FY-2001

    • Ethinyl Estradiol on Bone Growth in Rats

    • Erythromycin from Farmed Animals

    • Malachite Green/Leuco Malachite Green in Mice

    • Retinyl Palmitate: Isolation & Detection

    • DNA Adducts of Tamoxifen

    • Dietary Supplements & Herbals: Identification of Bioactive Ingredients


    Areas supported in fy 2001 continued

    Areas Supported in FY-2001(continued)

    • Endocrine Disrupters: Genistein & Daidzein

    • Phytoestrogen Conversion to Estrogenic Compounds: Genistein & Daidzein

    • Fluoroquinolone Biotransformation by Fungi

    • Microbial Degradation of Drugs & Feed Additives in Aquaculture

    • Antihistamine Drugs in Neonatal Mouse Cells


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