Forensic Anthropology and Isoscapes
Forensic Anthropology and Isoscapes
by
Department of Chemistry
Fall 2023
Oxygen isotope analysis in teeth: applications of isotope ratio mass spectrometry (IRMS) in
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
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
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
Keywords: Forensic anthropology, unidentified human remains, IRMS, tooth enamel, water
Forensic Anthropology
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
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,
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),
profile detailing origin, birthplace, residency, travel history, and diet can aid identification 5,
though obtaining this information becomes challenging after exhausting other leads.
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
3
geographical location. These characteristic chemical signatures, known as isotope fingerprints,
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
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
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
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
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
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
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
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,
by extracting and analyzing isotopes from biological tissues and pairing them with the isoscape
data5,16,17.
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
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
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.
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
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)
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 .
(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
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.
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.
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
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.
Isoscapes in forensic anthropology are crucial for law enforcement, providing strong
circumstantial evidence for investigative leads 7. The simplicity of this approach remains
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
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
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
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,
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
With almost every article I reviewed, I can say that I’m sure it will continue to expand and refine
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
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
19
References
21
(27) Ammer, S. T. M.; Bartelink, E. J.; Vollner, J. M.; Anderson, B. E.; Cunha, E. M. Spatial
Distributions of Oxygen Stable Isotope Ratios in Tap Water From Mexico for Region of
Origin Predictions of Unidentified Border Crossers. Journal of Forensic Sciences 2020, 65
(4), 1049–1055. https://doi.org/10.1111/1556-4029.14283.
(28) 12 state police officers charged after 19 bodies found near Mexico-U.S. border - CBS
News. https://www.cbsnews.com/news/12-state-police-officers-charged-killing-19-people-
mexico-us-border/ (accessed 2023-12-01).
(29) Masacre de Tamaulipas: declaran culpables a 12 policías de “fusilar y carbonizar” a 17
migrantes en el norte de México. BBC News Mundo.
https://www.bbc.com/mundo/articles/c0j3y858527o (accessed 2023-12-01).
(30) Laffoon, J. E.; Sonnemann, T. F.; Shafie, T.; Hofman, C. L.; Brandes, U.; Davies, G. R.
Investigating Human Geographic Origins Using Dual-Isotope (87Sr/86Sr, δ18O) Assignment
Approaches. PLOS ONE 2017, 12 (2), e0172562.
https://doi.org/10.1371/journal.pone.0172562.
(31) Martín-Gómez, P.; Barbeta, A.; Voltas, J.; Peñuelas, J.; Dennis, K.; Palacio, S.; Dawson,
T. E.; Ferrio, J. P. Isotope-Ratio Infrared Spectroscopy: A Reliable Tool for the Investigation
of Plant-Water Sources? New Phytologist 2015, 207 (3), 914–927.
https://doi.org/10.1111/nph.13376.
22