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Forensic Anthropology and Isoscapes

This document discusses using isotope ratio mass spectrometry to analyze oxygen isotopes in tooth enamel to determine the geographic origin of unidentified human remains. IRMS leverages location-dependent variations in heavy and light water isotopes influenced by climate. Integrated with GIS, this method creates isoscapes, maps showing isotope ratio distributions, which can reveal insights into the unknown region of origin.
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0% found this document useful (0 votes)
34 views23 pages

Forensic Anthropology and Isoscapes

This document discusses using isotope ratio mass spectrometry to analyze oxygen isotopes in tooth enamel to determine the geographic origin of unidentified human remains. IRMS leverages location-dependent variations in heavy and light water isotopes influenced by climate. Integrated with GIS, this method creates isoscapes, maps showing isotope ratio distributions, which can reveal insights into the unknown region of origin.
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Oxygen isotope analysis in teeth: applications of isotope ratio mass spectrometry (IRMS) in

forensic anthropology for geo-localization of human remains

by

Jaser Lara de Larrea

BSc, BEng, MASc, PhD Student

Department of Chemistry

CHEM 5206: Bioanalytical Mass Spectrometry

Professor: Dr. Alan Doucette

Fall 2023
Oxygen isotope analysis in teeth: applications of isotope ratio mass spectrometry (IRMS) in

forensic anthropology for geo-localization of human remains

Jaser Lara de Larrea

Abstract

In the late 1990s, an abundance of unknown human remains started to emerge along the

Mexican-US border as, sadly, immigrants were perishing trying to enter the US seeking better

opportunities. In normal circumstances, forensic anthropology can provide information using

skeletal examination to help identify the victims. However, with undocumented people,

traditional antemortem data (DNA, biometrics, medical records) are unavailable. As the only

possible solution isotope ratio mass spectrometry (IRMS) is explored to analyze tooth enamel

water isotopes (δ18O) to determine origin. IRMS leverages location-dependent variations in

heavy and light water isotopes, influenced by climate. Integrated with geographical information

systems (GIS), this method creates isoscapes, maps isotope ratio distributions, which is the key

for region-of-origin profiling. Despite its limitations, this approach can reveal insights into the

unknown region of origin, provide strong circumstantial evidence and establish the personal

history encoded in the isotopes of these undocumented victims.

Keywords: Forensic anthropology, unidentified human remains, IRMS, tooth enamel, water

isotopes, δ18O, isoscapes, geographic profiling.

Forensic Anthropology

Forensic anthropology is a specialized field within physical anthropology that focuses on

identifying human remains using skeletal analysis and archaeological methods in a legal setting 1.

1
It proves invaluable in medico-legal investigations when remains are not recognizable anymore

because of advanced decomposition, burning, drowning, scavenging, dismemberment,

mutilation, mummification, or skeletal conditions1. Obtaining a complete skeleton is rare in a

forensic context, often involving limited bones or fragments with potential degradation. Despite

these challenges, forensic anthropology techniques can still yield valuable information 2.

Forensic anthropology employs bone examination to create biological and trauma profiles

(Figure 1) as part of postmortem data (PMD)2,3. The biological profile includes estimating the

biological sex, height, age, and population affinity. To enhance specificity, the trauma profile

assesses antemortem, perimortem, or postmortem injuries. Old injuries provide insights into

health and habits, while the mechanism of injury aids in determining the potential murder

weapon and cause of death. Ultimately, the manner of death is classified as a homicide, suicide,

accidental, natural, or undetermined in forensic settings4.

2
Figure 1. Main profiles obtained by forensic anthropology techniques

While biological and trauma profiles offer insights into events surrounding the victim, they only

narrow down the search for identity. Standard identification techniques involve reviewing

antemortem data (AMD), including biometrics, biological data, and radiology reports. Cross-

matching PMD with AMD in a database is the next step 3. Still, identification becomes

challenging without AMD, particularly in cases predating the digital era (around the 1970s),

involving natural disasters, or with undocumented immigrants. In such cases, a background

profile detailing origin, birthplace, residency, travel history, and diet can aid identification 5,

though obtaining this information becomes challenging after exhausting other leads.

Isotope fingerprinting for geo-localization

Given that it could be the only viable option, stable isotope analysis proves beneficial for

background profiling6. Stable heavy and light isotopes of an element, characterized by their non-

radioactive nature, remain unchanged and do not undergo decay over time. In a biological

system, isotopes of a given element will interact chemically similarly; however, their atomic

mass differences will make them behave subtly differently physically. Heavier isotopes require

more energy to form and break bonds and react more slowly than lighter isotopes; natural

processes tend to favour the efficiency of the light isotope. This results in a difference in the

global natural abundance of isotopes and, therefore, a distinctive abundance within the different

components of the ecosystem. Stable isotope analysis is based on the principle that you are

where you live7. Isotopes within biological materials exhibit a distinct chemical signature that

appears as specific concentrations of elements such as C, H, N, O, and S. This distinctiveness

arises from biogeochemical processes depending on the environmental conditions and

3
geographical location. These characteristic chemical signatures, known as isotope fingerprints,

are traceable, which is helpful for geo-localization purposes.

Stable isotope analysis can be performed via isotope ratio mass spectrometry (IRMS). This

impactful technology has served in precisely measuring stable isotope composition in different

fields and has found insightful applications in forensic contexts 6. This technology has been

applied to trace seized drugs, determine the origin of explosives, food forensics (fake vs

authentic products) and wildlife forensics (illegal harvesting) 8. In forensic anthropology, isotope

analysis of biological tissues such as bones, nails, hair, and teeth has emerged as a method for

determining human provenance and narrowing down regions of origin 5. Although isotope

analysis from tissues cannot precisely identify the origin, it can eliminate numerous possible

search areas and concentrate efforts on ongoing investigations as an ultimate resource 9. Every

investigative lead toward obtaining more information is worth exploring in forensics because it is

better than having no leads.

Isotope Ratio Mass Spectrometry

Basic Principles

In nature, the biological components of an ecosystem will show different concentrations or ratios

(R) of the stable isotopes of bio-elements, such as C, H, O, N, and S. These ratios (R) are

described from a rare to an abundant isotope; for example, R= 13C/12C or R= 18O/16O. The delta

(δ) notation describes the slight deviations in the isotope ratio compared to a standard for light

stable isotopes, expressed in parts per thousand6. This is shown in equation 1:

18
δ O=
( R sample
Rstandard )
−1 ×1,000 (%) (1)

4
where Rsample and Rstandard are the 18O/16O ratios for a sample and the standard, respectively. The

isotope delta (δ), represented by the ratio of both measurements, clearly expresses slight isotopic

differences.

Isotopic fractionation

Heavy and light isotopes can be differentiated in natural (physical or biological) processes.

Isotopic fractionation is the partitioning of the isotopes by their mass (i.e., heavy and light

isotopes) between two phases. In water molecules, two stable isotopes of hydrogen (H 1 and H2,

often denoted by D, for deuterium) and oxygen (O16 and O18) give different isotopic

combinations. Heavier water isotopes form stronger bonds. The resulting heavy molecule tends

to dissociate less; thus, more energy is needed to break the intermolecular bonds. The increased

energy required to break the bond results in a slower reaction rate; therefore, natural processes

tend to favour the fractionation of lighter water isotopes 5. The water cycle is briefly explored as

an example of isotopic fractionation in nature.

5
Figure 2. Motility of δ18O in the water cycle. Heavier isotopes tend to be more abundant in low, warm regions and lighter
isotopes in high, cold areas. The climate (i.e., continentality, seasonality and altitude) mainly influences this isotopic
fractionation.

Natural isotopic fractionation processes occur throughout the water cycle, involving evaporation,

condensation, moisture transport, and diffusion (Figure 2). These processes are influenced by

climate factors such as continentality (distance from the coast), seasonality (temperature), and

altitude (elevation relative to sea level), leading to variations in isotopic compositions of drinking

water based on geographical location10. Water molecules with different isotopic compositions of

H and O are affected at each cycle stage. Heavier isotopes tend to remain in liquid form in

coastal areas due to their slower evaporation rate than lighter isotopes. Equilibrium-based

fractionation characterizes condensation and precipitation over land, while evaporation

undergoes kinetic or diffusion isotope fractionation, influenced by factors like wind speed and

temperature11. The global correlation between δ18O and δ2H in precipitation is established by

Dansgaard's equation (Equation 2):

2 18
δ H =s∗δ O+ d (2)

where 's' denotes the slope, 'd' represents the intercept, and the equation defines the Global

Meteoric Water Line (GMWL). This equation has been used in climatic studies to understand

better and compare past and present precipitation formation processes and, therefore, variation of

δ18O and δ2H values in different bodies of water 12. In equilibrium, the δ18O ratio is roughly 10%

higher in the condensed phase (liquid or solid) compared to the vapour phase. Within the cycle,

water undergoes significant atmospheric fractionation during the overall movement of vapour

(clouds) and precipitation across different latitudes (from low to high), ocean to continents, or

6
lower to higher elevations, owing to successive evaporation-condensation-precipitation

processes11.

For example, in North America (Figure 3), depending upon latitude, altitude, temperature and

distance to the seas, observed δ18O values precipitation water and, hence, fresh water can range

from -5.1 to -32.5% across the continent with the ‘heavier’ or less negative δ 18O values being

typical of warmer coastal near-equatorial regions and the ‘lighter’ or more negative δ 18O values

being typical of colder inland high latitude regions.

Figure 3. North America δ18O isotope distribution (isoscape) from annual precipitation13.

As a result of the different processes of isotopic fractionation in the water cycle, various isotopic

compositions of O tend to vary significantly in the geographical space, giving a unique

“fingerprint” on where to geolocate a specific isotopic abundance6.

7
‘Isoscapes’: applications for forensic anthropology

Isoscapes, when coupled with isotope data, serve as a tracking tool. They are geological maps

illustrating isotope ratio distribution, and they find applications in diverse fields such as bio-

archeology, ecology, oceanography, and hydrology. They play a crucial role in determining the

origin of fossils, tracking wildlife diets, and mapping precipitation distribution 14. Incorporating

isotope signatures and elemental models within geographical information systems (GIS),

isoscapes allow for depicting isotopic patterns and geo-localizing them in a specific geographical

space15. The isoscape in Figure 3 illustrates the distribution of δ 18O isotopes across North

America. It categorizes regions based on climate and each area's distinct δ 18O isotopic values.

This segmentation enables a focused examination of the specific regions depending on the

desired δ18O value. Consequently, individuals residing in the Mexican peninsula, for instance,

would exhibit higher δ18O values than those in Alaska. This distinction helps pinpoint a specific

region of origin based on the corresponding δ18O value. As shown in the existing literature,

isoscapes in forensic anthropology gained traction by helping identify region-of-origin profiles

by extracting and analyzing isotopes from biological tissues and pairing them with the isoscape

data5,16,17.

Tissues for isotope analysis

Throughout life, isotopes are absorbed within the body principally via ingesting food and water

from a determined geographical place. In forensic anthropology, tissues like teeth, bones, hair,

nails (keratin), and bone collagen are analyzed based on specific isotopes in specific tissues to

determine human provenance. Trace metal elements (Sr and Pb) indicating calcium substitution

are typically found in teeth and bones and analyzed by multicollection-inductively coupled

plasma mass spectrometry. Bio-elements like C, N, and S (dietary elements) in bone collagen,
8
hair, and nails, along with water indicators (H and O) identified in hair, nails (keratin), and teeth,

are analyzed through continuous flow isotope ratio mass spectrometry (CF-IRMS). The choice of

tissue is tailored to the forensic case, offering distinctive insights based on the presence of trace

metal and bio-elements.

For determining human geographic provenance (birthplace), tooth enamel is often analyzed for

isotope ratios of δ2H, δ18O, and 86Sr/87Sr 16. Teeth undergo a developmental process that spans

from pre-birth to the teenage years, during which they absorb chemical elements from the

individual's diet and water intake. Once teeth are formed, they do not undergo any further

changes18. Unlike bones, teeth lack regenerative capabilities after formation and are highly

resistant to degradation. The unchanging chemical composition of teeth preserves a childhood

environmental signature, offering a unique historical record19.

In protein digestion, amino acid oxygen isotopes mirror the gut water influenced by ingested

drinking water. Hydrogen atoms in amino acids undergo similar exchange at the C-terminus of

amino acids during protein degradation, but R-group hydrogen retains its original values.

Therefore, oxygen isotopes in unidentified decedent investigations only indicate iso-regions. In

contrast, hydrogen isotopes show a combined signal of dietary and iso-regional influences 20.

Tooth enamel minerals, comprising carbonate and phosphate, reflect the isotopic composition of

water an organism consumes9. In humans, the influence of food on oxygen isotope ratios in these

minerals is minor compared to drinking water. Therefore, a strong connection exists between the

O isotope ratios of enamel minerals and the water ingested by an individual. Due to its

efficiency, the measurement of carbonate δ18O values is prioritized over phosphate14.

9
Instrumentation and analytical technique

The measurement of the δ18O for tooth enamel can be performed with IRMS, which involves

four main steps. (1) The sample is transformed into simple gases; (2) introduction of the gases to

the ion source via the continuous flow (CF) interface; (3) ionization of the molecules with EI

followed by separation using a magnetic sector and detection of the ions in the mass

spectrometer; and (4) analysis of raw data. The amount of sample of tooth enamel is determined

by Formula 321:

1 mg 100
mg of sample needed for IRMS =µg of element needed ×( )×( )
1000 µg wt . % of theelement ∈ sample

(3)

Elemental Analyser (EA) and High Thermal Conversion (HTC)

The choice of combustion method is determined by the specific isotope being analyzed. In an

elemental analyzer (EA) coupled with a continuous flow (CF) system, Helium (He) is used as the

carrier gas injected into the EA inlet with the sample (). The EA transforms C, N, and S (bio-

appetite elements) into simple gases (CO 2, N2, and SO2). Samples, enclosed in a tin (Sn) capsule,

undergo combustion in the oxidation reactor where a pulse of oxygen (O 2) is injected (Sn + O2 →

SnO2 + heat), raising the temperature to 1020-1050°C. The sample gases, H 2O, NOX, unused

oxygen, and halides are produced from combustion. Halides are removed by a silver cobalt oxide

bed, followed by passage through a 650°C reduction reactor with copper wires to eliminate

excess oxygen and convert NOX to N2. After passing through a water trap to remove moisture,

the gases undergo gas chromatography (GC) to separate CO 2, N2, and SO2 before reaching the

CF interface.

10
11
Figure 4. Schematic of
22
the coupled EA- IRMS overall process and components .

Specifically for analyzing water isotopes, quantitative high-temperature conversion

(HTC) is used, an innovative technique that converts oxygen into CO and hydrogen to H 2 and

works similarly to the EA (Figure 5). This rapid and quantitative process occurs in a reducing

environment at temperatures reaching 1800˚C. The reactor, made of a glassy carbon tube,

ensures that neither the sample nor reaction gases come into contact with oxygen-containing

surfaces at high temperatures. This technology is unique in enabling memory-free conversion

reactions without restrictions on compound type. This chamber facilitates the conversion reaction

and separates reaction gases using a GC wire before they enter the CF interface.

12
Figure 5. Schematic of the HTC-IRMS overall process and components23.

Continuous Flow (CF) chamber

The gases then enter the IRMS through the CF interface (See Component 2 in Figures 4 and 5).

The CF allows for automatic sample gas dilution and the generation of reference gas pulses,

enabling individual referencing of each sample gas peak. The reference gas pulses can be

automatically adjusted in signal intensity, facilitating fully automated factor determination at any

point within a sample sequence. Here, sample and reference gases are transferred to the IRMS

for analysis.

Isotope Ratio Mass Spectrometer (IRMS)

The inlet of the IRMS (Error: Reference source not found) coupled with the combustion system

would be referred to as either EA-IRMS or HTC-IRMS. Within the source, molecules undergo

Electron Ionization (EI) with high-energy electrons (70 eV). The ions travel through the flight

tube, guided by a high voltage for focusing and acceleration 24. In the magnetic sector, ions of

different mass-to-charge ratios (m/z) follow a shared path through the magnetic field, one after

13
the other, yielding a mass spectrum with each scan. Ions in the magnetic field follow a curved

path for detection, influenced by balanced Lorentz and centrifugal forces. Adjusting magnetic

flux (B) or voltage (V) allows a detector at a specific radius to identify ions of any m/z. This ion

transmission and scanning mode define the magnetic sector mass analyzer. Its enduring

importance stems from high sensitivity for ultra-trace elements, simultaneous measurement of

multiple isotopes for precise ratios, and high-resolution capabilities to resolve spectral

interferences. Before high-performance alternatives emerged, such as time-of-flight (TOF) MS

and ion-cyclotron-resonance (ICR) MS systems, the magnetic sector was the only MS for such

high-resolution measurements and still serves today's needed purposes 25. At the end of their

trajectory, the ions are captured on Faraday cups. The Faraday cup is a piece of metal that resides

in the mass spectrometer's vacuum chamber and is connected to the instrument's electronics.

Upon striking the metal, ions are neutralized as electrons flow through the circuit to the Faraday

cup's surface. These detectors offer advantages for high-precision measurements based solely on

charge, avoiding mass discrimination. Additionally, ions with higher charge states produce

proportionally larger signals, facilitating the calculation of heavy-to-light isotope ratios. Multiple

cups enable simultaneous isotope measurements, compensating for minor overall ion beam

intensity fluctuations.26

14
Figure 6. Schematic of an isotope ratio mass spectrometer (IRMS). The image shows CO 2 entering through the inlet, getting
ionized by EI in the ion trap, and then individual ions moving through a flight tube. Then, a magnetic sector bends the trajectory
of ions based on their m/z, leading them to Faraday cups for detection.

Applications in Forensic Anthropology – Identification of US-Mexican undocumented

border crossers using IRMS (isoscapes)

Isoscapes in forensic anthropology are crucial for law enforcement, providing strong

circumstantial evidence for investigative leads 7. The simplicity of this approach remains

essential, amplifying its role in advancing forensic investigations, especially sensitive

humanitarian cases of deceased undocumented U.S.-Mexico border crossers27.

Since 1998, over 7,000 undocumented immigrants have died on the US-Mexican border 27.

Triggered by a severe economic recession in the mid-1990s, people sought better opportunities in

the US, driven by the allure of the "American dream." Faced with perceived complexities in the

US immigration process, many opted for illegal border crossings, viewing the risk as worthwhile

15
for a brighter future. The high death rates, especially in the western desertic zone near Arizona,

result from harsh environmental conditions, including extreme heat, wildlife dangers, and limited

resources. Ongoing violence along the border, involving local gangs, cartels, police, and the

army, further contributes to fatalities and the burning of remains 28,29. To address the challenge of

identifying numerous human remains, local authorities in Arizona explored the use of isotopic

analysis, specifically water isotopes, as a cost-effective and bioavailable method for determining

these remains' origins and facilitating their repatriation. The study addresses Mexico's lack of

isotopic reference data, specifically δ 18O in tap water. The authors created an isoscape mapping

δ18O tap water distribution across Mexico, analyzing 150 water samples nationwide to fill this

gap. Tooth enamel from unidentified remains (Individuals 1 and 2) was processed for isotopic

analysis through HTC-IRMS, and then isoscapes were generated using the ArcGIS software. The

isoscapes effectively pinpointed regions with specific isotopic values, successfully assigning

probable areas of origin (Error: Reference source not found). Upon positive identification,

Arizona authorities confirmed that the individuals' actual region of origin matched the predicted

isoscape regions.

The δ18O isoscape has proven to be a solid resource for region-of-origin prediction for these

recently identified undocumented border crossers 27. While demonstrating the isoscapes’ efficacy

in forensic identification, the study underscores the potential for future geolocation

advancements. The results suggest ongoing improvement and encourage further research into

analyzing multiple isotopes in different tissues and developing innovative isotopic methods. For

example, Laffoon et al. (2017) 30 proposed a dual isotope approach, utilizing isoscapes from
87
Sr/86Sr and δ18O in tooth enamel. Their study suggests the potential of refining regional

identification through overlapping regions. However, they highlight challenges in accessing Sr

16
isotope data due to bioavailability issues, in contrast to the more accessible nature of O isotopes.

Acknowledging these technological limits sets the stage for optimistic exploration and

refinement, indicating a promising future for this evolving forensic technology.

Figure 7. Isoscape map of tap water δ18O overlapped with the Individual’s 1 predicted region of origin (phosphorescent yellow
region). The actual region of origin from Individual 1 is highlighted with a blue star. Taken from Ammer et al., (2020).

As noted earlier, any tool or method that aids in uncovering investigative leads for identification

is worth exploring. The value lies in avoiding classifying the remains as “unknown.”,

underscoring the importance of examining every potential avenue in the quest for identification.

Although stable isotope analysis is still a developing technology, it is well-suited for forensic

investigations9.

17
Personal commentary.

In this review, I explored the application of IRSM in forensic anthropology. Despite being

considered an ‘old’ technology, IRMS's simplicity—a high-resolution and sensitivity mass

analyzer paired with simple and quantitative accurate detectors (faraday cups)—sets it apart from

other more modern technologies such as quadruples, TOF, or ICR MS. The practicality of IRMS

has proven outstanding in the forensic sciences8 Still, I believe that the main question to pose is,

“How impactful is this technology for forensic anthropology specifically?” This technology's

importance relies on providing clear investigative leads and strong circumstantial evidence to

narrow the search. Most importantly, identifying unknown decedents is and will remain a

fundamental aspect in all medico-legal investigations, and stable isotope analysis of human

tissues is the ultimate resource to give back their rights and humanity to the remains 9.

Recognizing that the remains under investigation represent real individuals who experienced

tragic fates is crucial. The duty is to exhaust all possibilities in reconstructing their identity,

seeking justice for what happened to them.

However, IRMS is not a perfect technology. The bulky magnetic sector, slow scan speeds, and

the need for high vacuum performance present challenges. Its novelty in forensic anthropology

cases has resulted in limited studies, with debates on the needed isotope profile—single or multi-

isotope. Concerns about isoscape data availability also arise, requiring specificity to both isotope

and tissue7. Despite challenges, IRMS gains traction in forensic anthropology within

investigative agencies, demonstrating growing recognition of its utility in human identification 9.

With almost every article I reviewed, I can say that I’m sure it will continue to expand and refine

as a technology well-grounded in chemistry and physics principles, with exciting opportunities

for stable isotope ratio analysis advancements in the near future. Exploring novel isotopes,

18
refining existing isoscapes, addressing postmortem alterations, and enhancing data reliability for

courtroom use is promising17.

Concluding remarks

IRMS technology has proven to be groundbreaking for many forensic applications. Although its

application has been relatively new in forensic anthropology, it holds immense significance by

providing valuable insights when traditional identification methods falter. Despite its unchanged

operation over decades, its simplicity, high resolution, and sensitivity endure.

The future of IRMs for forensic anthropology applications is still in development. The future of

IRMS in forensic anthropology is evolving, with challenges with standardized isotope profiles

and sample preparation. Isoscape technology faces constraints due to limited data on specific

isotopes from tissues and challenges with trace metal compound bioavailability. Moreover,

technologies like isotope ratio infrared spectroscopy, which are cheaper, might be a better

solution for analyzing isotopes, although spectral interferences of organic compounds limit their

use31. Despite its limitations, IRMS remains crucial, and its continual progress suggests a

substantial leap forward in the foreseeable future as more data emerges.

19
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