The Potential of Bulk and Amino-Acid Specific Isotope Ratio Mass Spectrometry of Human Hair in ...

155 Pages·2013·2.8 MB·English

by  Angela Mccutcheon

The Potential of Bulk and Amino-Acid Specific Isotope Ratio Mass Spectrometry of
Human Hair in Forensic and Clinical Applications

A dissertation presented to
the faculty of
the College of Arts and Sciences of Ohio University

In partial fulfillment
of the requirements for the degree
Doctor of Philosophy

Yan An
May 2013
© 2013 Yan An. All Rights Reserved.
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This dissertation titled
The Potential of Bulk and Amino-Acid Specific Isotope Ratio Mass Spectrometry of
Human Hair in Forensic and Clinical Applications

by
YAN AN

has been approved for
the Department of Chemistry and Biochemistry
and the College of Arts and Sciences by

Glen P. Jackson
Associate Professor of Chemistry and Biochemistry

Robert Frank
Dean, College of Arts and Sciences
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ABSTRACT
AN YAN, Ph.D., May 2013, Chemistry
The Potential of Bulk and Amino-Acid Specific Isotope Ratio Mass Spectrometry of
Human Hair in Forensic and Clinical Applications
Director of Dissertation: Glen P. Jackson
Isotope Ratio Mass Spectrometry (IRMS) is a specialized technique used to
measure the isotopic ratios at natural abundance of the non-radioactive isotopes, and has
proved to be a valuable tool in many applications. This research explores the use of
IRMS of human hair for clinical and forensic applications.
Several sample introduction methods are now available for commercial isotope
ratio mass spectrometers. Combustion is most commonly used for bulk isotopic analysis,
whereas gas and liquid chromatography are predominately used for the real-time isotopic
analysis of specific compounds within a mixture. The current state-of-the-art method to
study 13C/12C and 15N/14N isotopic ratio for a specific compound in complex biological
samples is still gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-
IRMS). However, because the volatility and the polarity of the compounds, as well as the
isotopic dilution and the kinetic isotopic effect, are complex issues for the GC separation,
an alternative LC separation method coupled to the IRMS device (LC-IRMS) is presented
in this work. Although the idea of combining the LC with isotope ratio mass
spectrometry was proposed over 10 years ago, the interface allowing LC-IRMS coupling
was only commercialized in 2004. This work indicates that currently more and more
developments based on LC separation coupled, among others, to IRMS mass
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spectrometers are performed to measure 13C isotopic ratio of non-volatile compounds.
We have started our experimental work by using the state-of-the-art method, GC-
C-IRMS, for the simultaneous analyses of ten amino acids form human hair. Through the
use of N,O-bis(trimethylsilyl) trifluoroacetamide (BSTFA) as a derivatizing agent, we
were able to separate and analyze ten of the more abundant amino acids in human hair.
EA-IRMS was used to analyze bulk isotope ratio of δ13C, δ15N and δ34S values of human
hair. Finally, we have applied LC-IRMS to measure amino acid in 20 different females
from Jordan between the ages of 17 and 50 years. The preliminary studies suggest that for
a relatively small sample size of 20 individuals, the subjects can be classified with a
reasonable degree of confidence into groups related to body mass index, age and dietary
habits. For leave-one-out cross-validated classification, cases were correctly assigned to
their groups 77% of the time for BMI and 83% of the time for broad age groups. These
time (length)-averaged amino-acid level δ13C analyses enable such classification
regardless of the short-term changes in each individual. The preliminary data warrant
more work to be conducted on a larger and more diverse sample size to better support
these initial observations.
As we show, IRMS is a highly discriminating technique for distinguishing
between hair samples from different human subjects. In the future, such biomarkers in
human hair could be used as an instrumental method of analysis for the statistical
comparison of known and questioned hair fibers, to provide physical or biometric
information about the donor of a questioned hair sample, or as a biomarker for diagnosis
in a clinical setting.
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DEDICATION

Dedicated to my mother and my father for giving their love and providing me with
everything and expecting nothing but happiness.
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ACKNOWLEDGMENTS
First of all, I would like to express my gratitude to my advisor, Dr. Glen P.
Jackson for giving me the opportunity of being a member in his research group. Not only
instructing me in my research, he also generously spent a lot of his precious time
reviewing and correcting my conference abstracts, papers and dissertation, helped me
practice my presentations. In addition, was it not for his far superior writing skills, this
dissertation would not have come to fruition. I would also like to thank my dissertation
committee, Dr. Shiyong Wu, Dr. Hao Chen, and Dr. Shigeru Okada, for agreeing to serve
on my dissertation committee. I also want to thank Dr. Glen P. Jackson for agreeing to
serve as the chair for my committee. I appreciate the time that they took to review my
dissertation and provide their learned expertise.
I would like to thank the Department of Chemistry and Biochemistry at Ohio
University for granting me the great opportunity of achieving my doctoral degree. The
Center for Intelligent Chemical Instrumentation is thanked for supporting conference
expenses. The West Virginia University is thanked for providing the last year research
assistant opportunity. I also want to acknowledge the financial assistance received from
the National Science Foundation grant number (0745590).
I also wish to thank my current lab mates, Bobby Deimler, Feng Jin, and former
lab mates Dr. Zeland Schwartz (ne Muccio), Dr. Shannon Cook, Dr. Carolyn
Zimmermann, Derrell Hood, Mengliang Zhang and Ayat Bani Rashaid. I would like to
thank Ayat in particular for collecting the hair samples and completed questionnaires in
Jordan and for sharing the samples and information. I would also like to thank the Chen
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group and the Harrington group, thank you for the companionship, helpful comments and
suggestions, you make my stay in the laboratory enjoyable. To all my friends, no matter
where you are, thank you for the friendship, this is always a precious asset in my life.
I must acknowledge the person who encouraged me to pursue my doctorate in the
first place, Dr. Tianlin Wang, my master degree research advisor at Shanghai University.
Dr. Wang saw the potential in me before I did and to him I will always be grateful.
Last but not least, I would like to thank my entire family, who has been my
grounding rock. I thank my father Yunqi An, my mother Limin Zhang, without your
constant support and encouragement I could not make this far. Although we are
separated by distance, I can feel your love every day. Each and every one of my family
has contributed to this accomplishment through encouragement, support, love, and
understanding. Without them I would not be the person I am today and my world would
not be as bright.
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TABLE OF CONTENTS
Page

Abstract ............................................................................................................................... 3
Dedication ........................................................................................................................... 5
Acknowledgments............................................................................................................... 6
List of Tables .................................................................................................................... 12
List of Figures ................................................................................................................... 13
Chapter 1: Introduction ........................................................................................... 19
1.1 Goals ..................................................................................................................19
1.2 Project Overview ...............................................................................................19
1.3 Isotope Ratio Mass Spectrometry ......................................................................22
1.3.1 EA-IRMS .......................................................................................................28
1.3.2 GC-IRMS .......................................................................................................30
1.3.3 LC-IRMS .......................................................................................................31
1.4 Application .........................................................................................................34
1.4.1 Forensic ..........................................................................................................35
1.4.1.1 Drugs ......................................................................................................35
1.4.1.1.1 Cocaine ..............................................................................................35
1.4.1.1.2 Marijuana ...........................................................................................37
1.4.1.1.3 Heroin and Morphine .........................................................................38
1.4.1.1.4 Synthetic Drugs ..................................................................................39
1.4.1.2 Explosives ..............................................................................................41
1.4.1.3 Wildlife Forensics ..................................................................................42
1.4.1.4 Environmental Forensics .......................................................................43
1.4.1.5 Food Authenticity ..................................................................................44
1.4.1.6 Paint .......................................................................................................47
1.4.2 Clinical ...........................................................................................................47
1.4.2.1 Nutrition .................................................................................................47
1.4.2.2 Metabolism ............................................................................................47
Chapter 2: isotopic analysis of human Hair ............................................................ 49
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2.1 Introduction ........................................................................................................49
2.2 Hair is an ideal medium for isotopic analysis ....................................................49
2.3 Measurement Methods of Hair Isotope Analysis ..............................................52
2.3.1 EA- IRMS ......................................................................................................52
2.3.2 GC-IRMS .......................................................................................................53
2.3.3 LC-IRMS .......................................................................................................53
2.3.3.1 Moving wire interface ............................................................................54
2.3.3.2 Chemical reaction interface ...................................................................55
2.3.3.3 Wet oxidation interface ..........................................................................57
2.3.4 ICP-MS ..........................................................................................................59
2.4 Applications .......................................................................................................62
2.4.1 Food and nutrition ..........................................................................................62
2.4.2 Diagnostic tool in disease ..............................................................................63
2.4.3 Human identification ......................................................................................64
2.5 Conclusions ........................................................................................................66
Chapter 3: 13C analysis of amino acids in human hair using trimethylsilyl
derivatives and gas chromatography-combustion-isotope ratio mass spectrometry ......... 67
3.1 Introduction ........................................................................................................67
3.2 Experimental ......................................................................................................72
3.2.1 Subject............................................................................................................72
3.2.2 Reagents and Standards .................................................................................72
3.2.3 Derivatization of Hair Samples ......................................................................73
3.2.4 Bulk Isotope Ratio Mass Spectrometry .........................................................73
3.2.5 Gas Chromatography – Mass Spectrometry / Isotope Ratio Mass
Spectrometry ..............................................................................................................74
3.3 Results and Discussion ......................................................................................77
3.3.1 Study of amino acid derivatization reaction conditions .................................77
3.3.2 Precision of isotopic measurement ................................................................79
3.3.3 Kinetic Isotope Effect (KIE) ..........................................................................82
3.3.4 Application of the BSTFA derivatization method to human hair
amino acids ................................................................................................................84
3.4 Conclusion .........................................................................................................85
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Chapter 4: Discriminating between human hair from different donors: A
preliminary assessment of the within-person and between-person Variance for
different IRMS capabilities ............................................................................................... 87
4.1 Introduction ........................................................................................................87
4.2 Experimental ......................................................................................................89
4.2.1 Subjects ..........................................................................................................89
4.2.2 Reagents and Standards .................................................................................89
4.2.3 Derivatization of Hair Samples ......................................................................90
4.2.4 Bulk Isotope Ratio Mass Spectrometry .........................................................91
4.2.5 Gas Chromatography – Mass Spectrometry / Isotope Ratio Mass
Spectrometry ..............................................................................................................92
4.3 Results and Discussion ......................................................................................94
4.3.1 Bulk isotope ratio values ................................................................................94
4.3.2 ICP-MS ........................................................................................................102
4.3.3 Compound specific isotope ratio values ......................................................104
4.4 Conclusion .......................................................................................................112
Chapter 5: EA-IRMS versus LC-IRMS amino-acid-specific isotopic
analysis of human hair .................................................................................................... 114
5.1 Introduction ......................................................................................................114
5.2 Experimental ....................................................................................................116
5.2.1 Reagents .......................................................................................................116
5.2.2 Hair Samples ................................................................................................116
5.2.3 EA-IRMS .....................................................................................................118
5.2.4 LC-IRMS .....................................................................................................119
5.3 Results and Discussion ....................................................................................121
5.3.1 Precision of EA-IRMS isotopic measurement .............................................121
5.3.2 EA-IRMS results of 20 human hair samples ...............................................122
5.3.3 Precision of LC-IRMS isotopic measurement .............................................125
5.3.4 LC-IRMS analysis of amino acid standards ................................................127
5.3.5 LC-IRMS analysis of human hair samples ..................................................130
5.4 Conclusion .......................................................................................................136
Chapter 6: Conclusions and Future Work ............................................................ 137