DTIC ADA421251: Solid Phase Microextraction (SPME) as a Method to Defect the Presence of Escherichia Coli in Waster by Headspace Sampling and Gas Chromatography/Mass Spectrometry Analysis

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1. REPORT DATE 2. REPORT TYPE 3. DATES COVERED
2002 N/A -
4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER
Application of Headspace Solid Phase Microextraction and Gas
5b. GRANT NUMBER
Chromatography/Mass Spectrometry for Detection of Escherichia Coli in
Water 5c. PROGRAM ELEMENT NUMBER
6. AUTHOR(S) 5d. PROJECT NUMBER
Tara L. Hall
5e. TASK NUMBER
5f. WORK UNIT NUMBER
7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION
Uniformed Services University of the Health Sciences REPORT NUMBER
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Approved for public release, distribution unlimited
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14. ABSTRACT
A method was developed for detecting the metabolite indole produced by Escherichia coli in the headspace
above a sample in Luria bertani broth. This method used solid phase microextraction (SPME) and gas
chromatography/mass spectrometry to perform extraction and analysis. Five commercially available
SPME fibers were investigated to determine the optimal fiber and extraction conditions. Polyacrylate (PA)
and polydimethylsiloxane-divinylbenzene fiber coatings gave a statistically indistinguishable and best
response compared to the other three types examined. The PA fiber coating was selected for further study.
The lower level of detection was determined to be 1 colony forming unit (cfu) in 100 milliliters (mL) of
water incubated for twelve hours at 37 degrees Celsius. Themethod detection level was determined to be
101-1000 cfu in 100 mL of water, although 11-100 cfu were detected 90.9% of the time. Forty water
samples split, filtered and analyzed with SPME and membrane filtration were shown to be in agreement
57.1% more often than would be expected by chance (Kappa = 0.571, P<0.001). The results of this study
showed significant promise in the use of SPME for detection of volatile metabolites of bacterial water
contaminants. However, under the conditions optimized in this study, the method is neither sensitive nor
fast enough to be considered superior over existing methods.
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manuscript entitled:

Solid Phase Microextraction (SPME) as a Method to
Detect the Presence of Escherichia coli in Water by
Headspace Sampling and Gas Chromatography/Mass
Spectrometry Analysis

beyond brief excerpts is with the permission of the copyright owner, and will save
and hold harmless the Uniformed Services University of the Health Sciences
from any damage, which may arise from such copyright violations.

Tara L. Hall
CPT, MS, U.S. Army
Department of Preventive Medicine and
Biometrics
Uniformed Services University of the Health
Sciences
i
ABSTRACT

Title of Thesis: “Solid Phase Microextraction (SPME) as a Method to Detect
the Presence of Escherichia coli in Water by Headspace
Sampling and Gas Chromatography/Mass Spectrometry
Analysis”

Author: CPT Tara L. Hall
Master of Science in Public Health

Thesis Directed by: CDR Philip A. Smith
Assistant Professor
Department of Preventive Medicine and Biometrics

A method was developed for detecting the metabolite indole produced by
Escherichia coli in the headspace above a sample in Luria bertani broth. This
method used solid phase microextraction (SPME) and gas chromatography/mass
spectrometry to perform extraction and analysis. Five commercially available
SPME fibers were investigated to determine the optimal fiber and extraction
conditions. Polyacrylate (PA) and polydimethylsiloxane-divinylbenzene fiber
coatings gave a statistically indistinguishable and best response compared to the
other three types examined. The PA fiber coating was selected for further study.
The lower level of detection was determined to be 1 colony forming unit (cfu) in
100 milliliters (mL) of water incubated for twelve hours at 37o Celsius. The
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method detection level was determined to be 101-1000 cfu in 100 mL of water,
although 11-100 cfu were detected 90.9% of the time. Forty water samples split,
filtered and analyzed with SPME and membrane filtration were shown to be in
agreement 57.1% more often than would be expected by chance (Kappa =
0.571, P < 0.001). The results of this study showed significant promise in the use
of SPME for detection of volatile metabolites of bacterial water contaminants.
However, under the conditions optimized in this study, the method is neither
sensitive nor fast enough to be considered superior over existing methods.
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APPLICATION OF HEADSPACE SOLID PHASE MICROEXTRACTION AND
GAS CHROMATOGRAPHY/MASS SPECTROMETRY FOR DETECTION OF
ESCHERICHIA COLI IN WATER

BY

CPT TARA L. HALL

Thesis submitted to the Faculty of the Department of Preventive Medicine and
Biometrics Graduate Program of the Uniformed Services University of the Health
Sciences in partial fulfillment of the requirement for the Degree of Master of
Science in Public Health, 2002
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DEDICATION
To my husband, Frank, for the sacrifices you have made during my Army
career and during the last two years of study. I dedicate this thesis to you and to
our two wonderful children, Kurt and Shyanne, who are the light, joy and
inspiration of my life.
To my mother Terry Glass, thank you for all your hard work and dedication
to me throughout my life. You taught me the value of hard work and
commitment, which has been paramount in completing this course of study.

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ACKNOWLEDGMENT
I am grateful to the Uniformed Services University’s Microbiology
Department for providing support to complete this work. I am especially grateful
to Dr. Michael Smith for providing the microbiology expertise to accomplish this
research.
I would like to thank all the members of my committee for their time and
assistance in helping complete this program. I also want to thank Cara Olsen for
her expertise in reviewing the statistical analyses performed and providing
guidance on sample size. I especially want to thank CDR Phil Smith and COL
(ret) Bob Fitz for giving me the idea for this research and encouraging me to stick
with it, even when the going was tough and I wanted to give up.
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TABLE OF CONTENTS

CHAPTER ONE: INTRODUCTION
Statement of Problem………………………………………………..…..……..1
Research Goal………………………………………………………..…………2
Research Question and Specific Aims .………………………….…………..3
CHAPTER TWO: LITERATURE REVIEW
Organisms……..………………………………………………………..……….4
Traditional Methods……………………………………………………..………5
Current Research……………………………………………………..…..…….7
Solid Phase Microextraction………………………………………..…….……7
Coatings………………………………………………………………...10
Why SPME?……………………………………………………………...….…12
CHAPTER THREE: MATERIALS AND METHODS
Overview………………………………………………………………………..14

Materials………………………………………………………………………..14
Gas Chromatography/Mass Spectrometry Methods……………………….15
Phase I…………………………………..…………………………………...…16
Phase II…………………………………………………………………………17
Quantitative Analysis of Indole in Solvent…………………….…….17
Initial Fiber Selection…………………………………………………..18
Effect of Sampling Time………………………………………………20
Broth Headspace SPME……………………….……………..………20
Broth Uptake…………………….……………………………………..21
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Lower Level of Detection……………………………………………...21
Method Detection Level………………………………………..……..22
Phase III…………………………………………………………….…………..24
CHAPTER FOUR: RESULTS AND DISCUSSION
Phase I…………………………………..……………………………………...26
Phase II…………………………………………………………..…….……….26
Quantitative Analysis of Indole in Solvent…………………………..26
Initial Fiber Selection…………………………………………………..27
Effect of Sampling Time…………………………………...……...…..27
Broth Headspace SPME……………………….……..……………....28
Broth Uptake…………………….……………………………….…….28
Lower Level of Detection……………………………………………...29
Method Detection Level……………………………………..….…….30
Phase III………………………………………………………………………...31
CHAPTER FIVE: CONCLUSION
Conclusions………………………………………………………………….....33
Limitations………………………………………………………………………34
Recommendations…………………………………………………………….35
APPENDIX…..…………………………………………………………………………45
BIBLIOGRAPHY…………………………………………………………….………...46
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