Stable Isotope Analysis: being included in books on forensic science [6, 7],

and finally a textbook dedicated to “stable isotope

General Principles and forensics” has been released [8].

Limitations
General Principles
What are Isotopes?
The basic building blocks of which we and every-
Introduction thing around us are made, i.e., the natural chemical
elements are listed in the periodic table of chemi-
The primary aims of forensic chemical analyses are cal elements in order of increasing atomic number
identification and comparison of various samples Z, which is the same as the number of protons in
with the intention of establishing a link between sam- the nucleus of an atom. Should two atoms of the
ples from a crime scene to samples relating to a same chemical element contain a different numbers
specific person or location (e.g., clandestine labora- of neutrons N in their nuclei, they are called isotopes.
tory). Identification, i.e., establishing chemical and Almost all of the 92 naturally occurring chemical ele-
structural identity by traditional methods of chemical ments are found in nature in more than one isotopic
analysis is often achieved through the characteriza- form. The vast majority of these isotopes are stable
tion of one or more specific sample constituents, isotopes, which means they do not decay. Radioiso-
while comparison often involves identification as topes by contrast are not stable and hence, undergo
well as quantification of multiple components in radioactive decay during which the parent element is
a sample of interest. With automated instrumenta- transformed into a lighter daughter element with a
tion widely available, highly specific spectroscopic lower atomic number than the parent element. In this
and mass spectrometry–based analytical techniques context “almost all” means with the exception of 21
are now, in most modern forensic science labora- elements such as fluorine and phosphorous, which are
tories, the most valuable tool used to achieve these monoisotopic.
analytical goals. The feature that characterizes a chemical element,
Should chromatographic and spectroscopic data which defines its chemical nature and makes, e.g.,
of two compounds correspond, it may be concluded carbon behave differently from sulfur is the number
that they are chemically indistinguishable. However, of protons in its nucleus since this is matched by
with increasing frequency an argument is brought for- the number of electrons surrounding the nucleus. The
ward by way of defence, asserting that samples that number of electrons and their quantum mechanical
are chemically indistinguishable may be chemically status define the nature and number of bonds a chem-
identical but are not necessarily the same. Being the ical element can form. A hydrogen atom contains one
same is interpreted here as sharing a common sam- proton and one electron, a carbon atom contains six
ple history or sample provenance. Multivariate stable protons and six electrons. The most abundant kind
isotope abundance analysis holds the potential to of carbon atom also contains six neutrons and this
resolve this argument by providing an additional set carbon species is called carbon-12 (12 C) since its
of independent variables (e.g., independent of other atomic mass is 12 atomic mass units or 12 amu. How-
spectroscopic characteristics) and thus increasing the ever, there is a less abundant form of carbon whose
overall discriminatory power of the forensic analy- nucleus contains still six protons but seven neutrons.
sis. Since the publication of the first articles applying Since the number of protons and, hence the num-
stable isotope analysis (SIA) to narcotic drugs [1, ber of electrons in this atom have not changed, the
2] thus providing proof of principle, advances and chemical characteristics of this atom are still those of
increasing interest in this emerging field of forensic carbon. However, since the additional neutron in its
sciences have resulted in an ever-increasing num- nucleus has increased its atomic weight by 1 from 12
ber of publications of original research to the point to 13 amu, it is called carbon-13 (13 C). So, in sum-
that in recent years the first case reports have been mary, isotopes of a given element contain the same
published [3–5], review articles on forensic SIA are number of protons (and electrons) and hence share the
2 Stable Isotope Analysis: General Principles and Limitations

same chemical characteristics but they contain differ- In the case of sulfur the mean isotope abundance
ent numbers of neutrons and are therefore of different figures for 32 S, 34 S, and 36 S are 95.02, 4.22, and 0.76
atomic mass. atom%, respectively. However, by using the above
global isotope abundance figures, one loses sight of
the variation in natural abundance of any given ele-
Stable Isotopes versus Radioisotopes
ment isotope. For 13 C, the range of this natural abun-
Isotopes that are not subject to radioactive decay but dance variation is of the order of 0.11 atom% [9].
persist in their same elemental form are stable iso- Approximately 75% or 0.0824 atom% of this vari-
topes. As everyday life experience tells us, almost all ation range falls within the interval 1.0343–1.1167
natural elements on Earth are stable isotopes. Since atom%. Changes in isotope abundance at natural
being an unstable isotope is an exception rather than abundance level cannot be reliably detected, let alone
the norm, radioactive isotopes have their own name, measured and quantified by analytical instruments
radioisotopes. Radioisotopes are not stable and will routinely used in a forensic chemistry laboratory.
decay over time, transforming in this process from Scanning organic desktop mass spectrometers (MS)
one element into another as parts of the nucleus leave equipped with a single electron multiplier detector
the atom during radioactive decay. Most radioiso- provide only an approximation of isotopic abundance
topes are either a product of nuclear fission (e.g., through the detection of isotope satellite ions, e.g.,
90
Sr, t1/2 = 28.8 y) or cosmic rays–induced reac- M+1+ and comparing its selected ion current with
tions in the upper atmosphere. The familiar example that for the corresponding major abundant fragment
of 14 C (carbon-14 dating) is the result of cosmic ion M+ [10]. Staying with the example of carbon and
rays–induced reactions in the upper layers of the tro- its minor isotope 13 C, in day-to-day practice, scan-
posphere and is yet another carbon isotope whose ning desktop MS systems can reliably detect changes
nucleus contains six protons and eight neutrons. How- in isotope abundance if a compound has been labeled
ever, unlike the other two stable isotopes of carbon, with 13 C, so the 13 C content of the compound is in
12
C and 13 C, the carbon isotope 14 C is not stable. It excess of 0.5 atom% above the typical natural abun-
is a radioisotope, which decays with a half-life t1/2 dance level of 1.11 atom%, i.e., containing >1.61
of 5730 ± 40 y. atom% of 13 C.
Given that isotope abundance is measured by
Stable Isotope Abundance and the δ-Notation instruments with dedicated channels per isotope (mul-
ticollector MS), expressing natural abundances of
Typically, global or mean figures for isotope abun- stable isotopes in a given material as the ratio of
dances of an element are given as atom percentages the minor (usually heavier) over the major abun-
(see Table 1). For 12 C and 13 C the figures quoted in dant (usually lighter) isotope of a given element (e.g.,
2
textbooks are 98.89 and 1.11 atom%, respectively. H/1 H or 13 C/12 C) has been adopted as the preferred

Table 1 Stable isotopes of light elements and their global natural abundance to two significant
decimals
Major First minor Second minor Isotope ratio
abundant abundant abundant first minor/major
Chemical isotope isotope isotope for scale calibration
element (at%) (at%) (at%) material
1 2
Hydrogen H 99.985 H 0.015 0.00015576 VSMOW
12 13
Carbon C 98.89 C 1.11 0.0112372 VPDB
14 15
Nitrogen N 99.63 N 0.37 0.0036765 AIR
16 18 17
Oxygen O 99.76 O 0.20 O 0.04 0.0020052 VSMOW
32 34 33
Sulfur S 95.02 S 4.22 S 0.76 0.0450045 VCDT
 Wiley-Blackwell 2010. Reproduced with permission from Meier-Augenstein 2010 [8]. VSMOW,
Vienna Standard Mean Ocean Water; VPDB, Vienna Pee Dee Belemnite; VCDT, Vienna Canyon Diablo
Troilite; at%, atom%
 Stable Isotope Analysis: General Principles and Limitations 3

notation. Since changes in isotope composition of three years. For day-to-day use it is therefore a com-
light elements at natural abundance level are rela- mon and accepted practice for laboratories to use
tively minute and isotope abundances of the minor tertiary comparison materials (in-house standards),
isotopes are very small, significant changes when which have been calibrated by the laboratory against
given in atom% happen typically in the second deci- international CMs and RMs. Laboratories are strongly
mal place. Similarly, changes in the ratio of heavier advised if not expected to share their in-house stan-
: lighter isotope are usually confined to the numerals dards with other laboratories so as to have δ val-
in the third and fourth decimal places. Owing to the ues assigned to these materials independently val-
minute nature of these changes in isotope abundance idated. Interlaboratory comparisons (ILCs) and so-
at natural abundance level, measured isotope ratios called round-robin tests are also a common and highly
of a given material are expressed relative to a con- recommended practice to assess proficiency and thus
temporaneously measured isotope ratio of a standard to ensure that analyses carried out in different labo-
of known isotopic composition. To make the result- ratories yield the same results.
ing figures more manageable, the “delta notation” (δ)
was adopted. The δ-value of the heavier isotope h Reference Scale Calibration
of a chemical element E in a sample is defined by
the following equation (equation (1)) [9, 11] where Stable isotope data for any light element stable iso-
Rsample is the measured isotope ratio of the heavier tope must be reported in the literature on the scale
isotope over the lighter (e.g., 13 C/12 C) for the sam- defined by the CM for corresponding isotope, e.g.,
13
ple and Rstandard is the measured isotope ratio for the C data are reported on the VPDB scale while 2 H
corresponding international reference material (RM) data are reported on the Vienna Standard Mean Ocean
(e.g., Vienna Pee Dee Belemnite, VPDB in the case Water (VSMOW) scale. All modern continuous flow
of 13 C). isotope ratio mass spectrometer (CF-IRMS) systems

   come equipped with an inlet permitting mass discrim-
Rsample ination free introduction of a suitable reference gas
δ E=
h
− 1 × 1000 (1)
Rstandard into the ion source. This approach safeguards against
day-to-day variation in the isotope ratio mass spec-
The results of this equation are referred to as per trometer (IRMS) performance but does not address
mil values (‰). A positive δ value is interpreted important issues such as the “principle of identi-
to mean the sample has a higher abundance of the cal treatment” (PIT) of sample and RMs for mean-
heavier isotope than the international reference stan- ingful correction and quality control of analytical
dard that defines the scale for a particular isotope, results [12–15]. Since the nature of bulk SIA by
while a negative δ value is interpreted to mean the elemental analyzer isotope ratio mass spectrometry
sample has a lower abundance of the heavier iso- (EA-IRMS) prevents us from mixing the sample with
tope than the international reference standard. By a standard, good practice demands bracketing a batch
virtue of its definition, the δ value of a scale defining of samples by a set of two standards of known, yet
international reference standard is 0‰. International different isotopic composition (ideally two interna-
standards that define the scale of a particular isotope tional RMs or CMs [8, 11, 16–20]). In addition, sys-
are called calibration materials (CM s), while inter- tem performance should be cross-checked by includ-
national standards whose isotopic composition have ing at least one RM or an in-house standard treated
been certified by direct comparison with the relevant as unknown sample as part of a batch run to act as an
CM are called reference materials (RMs). CMs and acquisition quality control sample. Typically, a batch
RMs are certified and administered through the Inter- analysis may consist of four or five actual samples
national Atomic Energy Agency (IAEA) in Vienna, run at least in triplicate with standards (in triplicate)
Austria, or the National Institute of Standards and on either side for subsequent end-member correction
Technology (NIST), Gaithersburg, USA. [8, 14].
Stocks of international CMs and RMs are finite Since deviations of measured from expected or
and hence, available only in limited quantities. Owing certified isotope abundance values of a given stan-
to this restriction, some of these materials can only dard are composed of a dynamic factor a (the scale
be reordered by laboratories engaged in SIA every compression or stretch factor) and an off-set b (or
4 Stable Isotope Analysis: General Principles and Limitations

shift), at least two reference points are required for 1.0817 and 1.0985 atom%, respectively, or more con-
scale calibration of measured δ-values to yield scale- veniently expressed in the δ-notation, the δ 13 C value
normalized δ-values that will permit intra- as well as of cane sugar is −11.59‰ and thus 15.28‰ more
interlaboratory comparison of data free of any instru- positive than the beet sugar δ 13 C value of −26.87‰.
ment or sample conversion bias. By treating measured In other words, two chemical compounds, indeed,
isotope abundance values as variable x and expected two molecules can be chemically identical but can
or certified isotope abundance values as a function of be readily distinguished by their isotopic composi-
x, a straightforward linear regression analysis of the tion. Why is that?
data for two RMs will yield a calibration equation of Even though two isotopes of the same chemi-
the form shown in equation (2). cal element have the same chemical characteristics,
this similarity ends the moment that chemical bonds
δ(expected) = a × δ(measured) + b (2) have been formed and we are looking at molecules,
chemical compounds. Once part of a molecule, the
For a two-point scale calibration to be meaningful, minute differences in atomic mass have quantifiable
the two RMs have to be chosen so as to envelope and measurable effects. Differences in the zero-point
the range of δ-values assumed by the samples. For energy of chemical bonds containing one heavy iso-
example, if a set of samples is expected to yield tope and one light isotope relative to bonds containing
δ 13 C-values ranging from −15‰ to −25‰, the two light isotopes are reflected by differences in
RMs should have δ 13 C-values of about −10‰ and the rates of cleavage of these bonds because dif-
−30‰, respectively. This type of scale calibration ferences in zero-point energy results in differences
is referred to as two-point end-member correction. in bond energy. For example, for hydrogen gas, the
For analysis of 13 C and 18 O isotope abundance in bond strengths of 1 H– 1 H, 1 H– 2 H, and 2 H– 2 H are
carbonates, the two recommended anchors to be used 436.0, 439.4, and 443.5 kJ mol−1 , respectively. Thus,
2
for end-member correction are carbonate minerals H– 2 H bonds contain more energy and are hence bro-
themselves, and are called NBS19 and LSVEC [16]. ken at a slower rate than 1 H– 2 H bonds, which in
turn are broken at a slower rate than 1 H– 1 H bonds.
The infrared (IR) spectra of carbon dioxide (CO2 ) are
How it Works another fine example of how differences in isotopic
makeup and therefore differences in bond length and
Why can Chemically Identical Compounds be bond strength result in differences in vibration energy
distinguished by their Isotopic Composition? states between neighboring IR absorption bands of
2310.0 and 2310.35 cm−1 for the stretching vibrations
While one could argue that the isotope abundances of 12 CO2 and 13 CO2 , respectively. While the differ-
of all elements present on earth were fixed when the ence in IR absorption bands for isotopologues such
Earth was formed and, on a global scale have not as 12 CO2 and 13 CO2 may only be measurable on a
changed since, the same is not true with regard to microscopic scale, for other compounds, differences
the various subsystems or microcosms in the global in their isotopic composition can lead to different
system that the Earth is composed of. Compartmental properties of macroscopic proportions. Water is such
isotope abundance of light elements is not fixed but a compound. Owing to its ability to form hydrogen-
is in a continuous state of flux due to mass discrim- bridging bonds, a kind of intermediate bond that
inatory effects of biological, biochemical, chemical, can either be dissolved or be converted into a cova-
and physical processes. For instance, when looking lent bond (at the expense of a prior covalent bond),
at individual carbon pools, one finds some with a its response, e.g., to a change in temperature (see
higher abundance of 13 C such as marine carbonate Table 2) is subject to a combination of both primary
sediments, whereas others are more depleted in 13 C and secondary isotope effects (see below).
such as hydrocarbons found in crude oil. Similarly, Considering the differences in bond length and,
when comparing the 13 C isotopic composition of cane hence, bond strength between bonds in a molecule
sugar one finds this to be different from the 13 C that involve different isotopes of the same chemical
isotopic composition of beet sugar. The 13 C isotope element already results in measurable differences in
abundance in beet sugar and cane sugar is on average spectroscopic characteristics, it should therefore come
 Stable Isotope Analysis: General Principles and Limitations 5

Table 2 Influence of isotopic composition on fixed point properties

of water
Property 1
H16
2 O
2
H16
2 O (D2 O)

Molar mass (g mol−1 ) 18.02 20.03

Maximum density (g cm−3 ) 0.99995 1.1053
Melting point at 101.325 kPa ( ° C) 0.00 3.82
Boiling point at 101.325 kPa ( ° C) 100.00 101.42

as no surprise that the same differences do also lead isotope effect [23] because it manifests itself in pro-
to different reaction rates when comparing a bond cesses where chemical bonds are neither broken nor
between the more abundant lighter isotopes of the formed. Typical examples for such processes in which
same or different chemical elements with a bond the results of thermodynamic isotope effects can be
involving one or, more so, two heavier isotopes [21]. observed are IR spectroscopy and any kind of two-
The most significant isotope effect is the kinetic or phase partitioning (e.g., liquid–liquid extraction) or
primary isotope effect, whereby a bond containing phase transition (e.g., liquid to gas, i.e., distillation
the chemical elements under consideration is either or vaporization). The thermodynamic isotope effect,
broken or formed in the rate-determining step of the or physicochemical isotope effect, is the reason for
reaction [22]. the isotopic fractionation observed during chromato-
The second type of isotope effect is associated graphic separations [24–26] and why the vaporiza-
with differences in physicochemical properties such tion of ocean surface water resulting in clouds (i.e.,
as IR absorption, molar volume, vapor pressure, boil- water vapor) being depleted in both 2 H and 18 O as
ing point, and melting point. Of course, these prop- compared to ocean surface water. Precipitation falling
erties are all linked to the same parameters as those from a cloud is in essence a reversal of this process.
mentioned for the kinetic isotope effect, i.e., bond Rain containing a higher proportion of the heavier
strength, bond length, and vibration energy levels. isotopes 2 H and 18 O precipitates, while the remain-
However, to set it apart from the kinetic isotope ing cloud becomes increasingly more depleted in 2 H
effect, this effect is referred to as thermodynamic and 18 O (see Figure 1).

Initial Later
precipitation precipitation
d18O = −12‰ d18O = −21.6‰
d2H = −84.4‰ d2H = −156.1‰

Vapor Vapor Vapor

d18O = −2.8‰ d18O = −12.4‰

d2H = −11.8‰ d2H = −89.2‰
Rain Rain
Evaporation

Ocean Continent
d18O= 0‰ The “continental” effect: repeated isotopic fractionation
d2H = 0‰ during a succession of separate precipitation events.

Figure 1 Isotopic fractionation during cloud formation and subsequent rainfall

6 Stable Isotope Analysis: General Principles and Limitations

Mass discrimination and resulting isotopic frac- information regarding variation in isotopic composi-
tionation that are associated with Rayleigh pro- tion of precipitation can be found at the hydrogeology
cesses [27] such as evaporation, condensation, and section of the IAEA http://www-naweb.iaea.org/napc/
precipitation of meteoric water ultimately result in ih/IHS resources gnip.html
source water having different isotopic composition It should be noted that the isotopic composition
depending on geo-location. Depending upon latitude, of precipitation is subject to seasonal variance in line
altitude, temperature, and distance to the open seas, with seasonal changes in temperature. However, the
observed δ 2 H-values of meteoric water (precipita- isotopic variance of water we consume be it directly
tion) and, hence freshwater can range from 20‰ or be it indirectly as an integral part of fruit and
vegetables is much less than the isotopic variance
to −230‰ across the world with the “heavier” or
of precipitation. Most tap water is either fed from
less negative δ 2 H-values being typical of coastal/near
groundwater sources (aquifers) or from freshwater
equatorial regions and the “lighter” or more negative reservoirs. Owing to mixing, these freshwater sup-
δ 2 H-values being typical of inland/high-altitude/high- plies exhibit a time-averaged stable isotope signature.
latitude regions. Geographical information system So, tap water supplied from a freshwater reservoir
(GIS) maps and contour maps of meteoric 2 H and will show a smaller seasonal isotopic variance than
18
O isotope abundance (cf. Figure 2) are in the pub- precipitation while the isotopic variance of tap water
lic domain and can be accessed via the Internet supplied from groundwater will be even less. Given
from resources such as http://www.waterisotopes.org, the slow recharge of aquifers, the isotopic signature
which is maintained by Gabriel Bowen at Pur- of groundwater represents a time-averaged signal of
due University [28, 29]. Another useful resource of hundreds of years.

d2H of Annual precipitation

6

to 8
11 12

−1 16
0
16

32
55 25

39 24

23 22

07 20

91 19

75 17

59 16

43 14

27 12

−9 −9

−7 −8

−6 −6

−4 −4

−3 −3

−
−1 −1

−
5
−2 o −

−2 o −

−2 o −

−2 o −

−1 o −

−1 o −

−1 o −

−1 o −

−1 o −

to

to

to

to

to

to

17
t

1
71
−2

Figure 2 Global isoscape of annually averaged 2 H isotope abundance in precipitation. [Reproduced with permission from
G.J. Bowen, Purdue University, IN, USA. http://wateriso.eas.purdue.edu/waterisotopes/index.html.  Gabriel J. Bowen.
http://wateriso.eas.purdue.edu/waterisotopes/index.html]
 Stable Isotope Analysis: General Principles and Limitations 7

Next to isotopic fractionation during evaporation this means the isotopic abundance of one carbon in
and precipitation of water, CO2 fixation in plants every molecule of a given organic compound has
during photosynthesis is one of the best studied and to increase either to 1.61 atom% (+0.5 APE) or to
documented consequences of mass discrimination decrease to 0.61 atom% (−0.5 APE) with neither
in nature. While the net isotopic fraction between value being anywhere near typical natural abundance
atmospheric bound carbon as CO2 (δ 13 C ≈ −8‰) levels for 13 C. In other words, single-collector scan-
and organic bound carbon in C3 -plants that use the ning MS cannot provide reliable quantitative informa-
Calvin–Benson cycle for photosynthesis amounts to tion on differences in isotopic composition of organic
∼ − 20‰, it amounts only to ∼ − 4‰ for organic compounds at natural abundance level caused by the
bound carbon in C4 -plants that use the Hatch–Slack kinetic or thermodynamic isotope effects mentioned
cycle for photosynthesis (see Figure 3) [30–32]. above. For this reason, multicollector isotope ratio
mass spectrometers (IRMS) have become the instru-
Measuring Stable Isotopic Composition ments of choice to determine origin or history of a
given organic compound by measuring its character-
Owing to reasons of instrument design already briefly istic isotope “signature” [23, 36, 37]. Common to all
mentioned above, under optimized conditions, the IRMS systems is the universal triple collector, but
achievable precision of isotope abundance measure- systems with five or even eight collectors are com-
ment using a molecular MS in selected ion mon- mercially available.
itoring (SIM) mode can only be as good as 0.05 IRMS are nonscanning magnetic sector instru-
atom% but realistically will be of the order of 0.1–0.5 ments in which mass-filtered ions are stigmatically
atom%. The resulting limitation on accuracy of iso- focused onto dedicated Faraday cup (FC) collectors,
tope abundance measurements in SIM mode therefore typically a triple collector array, positioned along the
only permits the detection of changes in isotopic focal plane of the full deflection radius r in such a
abundance of the order of 0.5 atom% excess (atom way that the each ion beam composed of ions of a
percentage excess, APE) [10, 33–35], e.g., in the particular mass (represented by the mass/charge ratio
case of 13 C (overall natural abundance 1.11 atom%) m/z) falls into its appropriate cup for continuous and

NP

60

30

EQ

−30

−60

SP
180 120 W 60 W 0 60 E 120 E 180
−27.7 −27.0 −26.3 −25.7 −25.0 −22.0 −19.0 −16.0 −13.0

−28.0 −27.3 −26.7 −26.0 −25.3 −23.5 −20.5 −17.5 −14.5

Figure 3 Global isoscape of mean annual δ 13 C values of plant carbon. [Reproduced with permission from Ref. 30. 
American Geophysical Union, 2005]
8 Stable Isotope Analysis: General Principles and Limitations

simultaneous ion recording of all ions and, hence gases before introduction into an IRMS, stable iso-
complete data integration. IRMS instruments achieve tope analysis can be applied to samples that consist
highly accurate and precise measurement of isotopic of a single compound or mixture of compounds (bulk
abundance, however, at the expense of the flexi- stable isotope analysis, BSIA), specific compounds
bility of scanning MS. IRMS systems have been within a complex sample such as an individual fatty
specifically designed to measure isotopic composition acid within a lipid sample (compound-specific iso-
at low-enrichment and natural-abundance levels by tope analysis, CSIA), and specific locations within a
simultaneous detection or ion counting of all minor complex molecule (position-specific isotope analysis,
and major abundant isotopologues. Since it is not fea- PSIA). The most commonly encountered analytical
sible to build an IRMS with a multicollector system technique is still BSIA. Samples are either mea-
covering all masses from 2 to 600 amu, for example, sured at the bulk material level, e.g., an ecstasy
any complex material or compound subject to SIA table without prior separation of its active ingredi-
has to be converted into a permanent gas such as ent 3,4-methylenedioxymethamphetamine (MDMA)
carbon dioxide (CO2 ) or nitrogen (N2 ) that must be from excipients (bulking or cutting agents), or at the
isotopically representative of the parent material. bulk compound level, e.g., MDMA extracted from
The sample gas generated by sample conversion an ecstasy tablet ([38], for more detailed information
at temperatures in excess of 900 ° C is subsequently on stable isotope signatures of narcotic drugs, please
introduced into the ion source of the IRMS, from refer to the article Stable Isotope Analysis: Drugs.
where molecular ions emerge that are accelerated into
the analyzer by a fixed accelerating voltage V , and are What Does a Stable Isotope “Signature” or
separated by a magnetic sector set to a fixed magnetic “Profile” Tell Us?
field strength B throughout the analysis. Unlike elec- While isotope fingerprint is already a commonly used
tron multipliers used in scanning organic MS where term in the isotope field to describe a distinguishing
a hit by a fragment ion leads to an electron cascade, combination of stable isotope abundances of different
FC detectors are actual ion counters. As standard, elements in a particular specimen, this term should
IRMS instruments are equipped with a triple cup col- be avoided, since, unlike an actual fingerprint, signa-
lector array composed of three FCs for measurement tures can and do change over time, and the same
of masses m/z 44, 45, and 46, for example, with applies to stable isotope signatures. For example,
each FC positioned in such a way that the ion beam the bulk isotopic composition of a crude oil sample
of each charged mass falls simultaneously into the can change over time as the more volatile, low-
appropriate cup. The masses m/z 44, 45, and 46 cor- boiling-point constituents evaporate. The terms stable
respond to the CO2 isotopologues 12 C16 O2 , 13 C16 O2 , isotope signature or stable isotope profiles are used to
and 12 C18 O16 O, respectively. describe a multivariate set or combination of δ values
High-precision SIA of 13 C isotopic abundance at for the minor isotopes of two or more of the chemi-
both natural-abundance and low-enrichment levels, cal elements present in a particular sample. Should a
for example, can yield measurements of 13 C abun- sufficient body of background knowledge exist, e.g.,
dance with a typical precision of ±0.0001 atom% in the form of databases or published research, it may
or ±0.1‰. Thanks to this high precision, even be possible to draw some generic conclusions from
minute changes in 13 C isotopic abundance of 0.0003 the stable isotope signature of a given material or
atom% or 0.25‰ can be reliably detected, irrespec- compound. At a more basic level, stable isotope sig-
tive of whether these minute changes have been natures can be used to compare a subsample taken
caused by kinetic isotope effects associated with from a particular exhibit with those from one or more
enzyme-mediated biochemical reactions or batch-to- other exhibits. The fact that sugar produced by dif-
batch variations of reaction conditions during its ferent plant species can differ significantly in its 13 C
chemical synthesis. isotopic composition is a good example to illustrate
The quality of the accuracy of the analytical pro- these points (see Table 3).
cess described above is controlled by contempora- Traditional analysis of different samples of caster
neous analysis of samples of known isotopic com- sugar will be able to confirm their chemical iden-
position. Since samples are being converted, e.g., tity as that of sugar but will not be able to dis-
combusted to yield isotopically representative simple tinguish between samples of different provenance.
 Stable Isotope Analysis: General Principles and Limitations 9

Table 3 Isotopic abundance of 13 C and 2 H in caster sugar from different plant

sources and of different geographic origin
Sample δ 13 CVPDB (‰) δ 2 HVSMOW (‰)
Caster sugar (sugar beet, Poland) −25.42 −71.0
Caster sugar (sugar beet, Sweden) −26.84 −93.4
Caster sugar (sugarcane, Brazil) −11.76 −21.4
Caster sugar (sugarcane, South Africa) −11.10 −6.7

Armed with the knowledge that C4 plants yield plant by differentiating between source and/or origin it can
products that relative to the international scale ref- be asserted that two substances, which do not share a
erence point VPDB contain more 13 C than plant common history, provenance, or relationship are not
products of C3 plants, bulk 13 C isotope analysis of truly identical even if they are chemically indistin-
caster sugar quickly reveals which product was made guishable. Stable isotope signatures or profiles can
from a C3 plant (e.g., sugar beet) or a C4 plant (e.g., contest this assertion. If a sample or substance of
sugar-cane). Additionally carried out bulk 2 H isotope unknown provenance appears to have the same multi-
analysis may reveal further differences between the variate stable isotope profile as a control sample, i.e.,
samples such as different geographic origin of the a control sample or substance of known source and
parent plant source. It is possible to interpret dif- origin, it is possible and quite likely for the unknown
ferences of observed δ 2 H values in a general way sample to share a common history or provenance with
by stating samples exhibiting more negative δ 2 H val- the control sample. Conversely, should multivariate
ues originated from regions of higher latitude, more stable isotope profiles of the unknown samples (e.g.,
distant to the coast, and a colder climate as com- suspect) and the control samples (e.g., crime scene)
pared to the lower latitude, more nearer the coast, differ, then it is likely they do not share a common
and warmer climate regions from which the samples history or provenance (see Figure 4).
with less negative δ 2 H values came from. However, In summary, a stable isotope signature or profile
any further interpretation beyond these points, i.e., can tell us whether two otherwise indistinguishable
of a positive predictive nature has to be backed up compounds are possibly related, i.e., share a common
either by comparative analysis of control samples history or provenance or not. In addition, they may
of known sample history and provenance or, bet- tell us something about origin and source of a
ter still, by a database covering samples from all compound by providing information about where a
sugar-farming regions collected repeatedly over sev- compound was made and by whom or by which
eral years. Analytical methods traditionally applied process.
in forensic science laboratories aim to establish a
degree of commonality between one substance, e.g.,
associated with the victim or the crime scene and Forensic Applications of Stable Isotope
another, e.g., associated with the suspect by identi- Analysis
fying its constituent elements, functional groups, and
What are the Key Aims for Stable Isotope
by elucidating its chemical structure. So should the
analysis of two colorless, crystalline samples result Signatures in a Forensic Context?
in identical mass spectra, IR spectra, melting points, In their recently published article “Stable Isotopes
etc., that are all consistent with published reference and the Courts”, Ehleringer and Matheson define
data for sugar, one can safely conclude two things: three key aims for stable isotope signatures or profiles
(i) both samples are chemically indistinguishable and in a forensic context [39].
(ii) both samples are comprised of sugar. However, it
can be argued that although two substances in ques- 1. The first aim is to ascertain whether two seem-
tion are chemically indistinguishable they may yet ingly indistinguishable specimens also share the
not be the same since they may have come from dif- same isotopic composition and may therefore be
ferent sources (producers) or be of different origin related, e.g., whether they share a common his-
(geographic location of production). In other words, tory or provenance. This application describes
10 Stable Isotope Analysis: General Principles and Limitations

−50.00

−60.00

−70.00

−80.00
d2HVSMOW (‰)

−90.00
Bopeep X
−100.00 Swan Vesta A
Swedish
−110.00 Swift
Crime scene
Crime scene
−120.00
Suspect
Suspect
−130.00 Spar
Chavi
−140.00
−30.00 −29.50 −29.00 −28.50 −28.00 −27.50 −27.00 −26.50 −26.00 −25.50 −25.00
d13CVPDB (‰)

Figure 4 Bivariate 2 H/13 C stable isotope profile of wooden safety matchsticks. Symbols representing samples collected
from the crime scene and seized from the suspect are encircled dark red and light blue, respectively. All other symbols
represent control matchstick samples of known provenance

the classic forensic identification task of com- 3. The third aim is to determine a probable geo-
paring an unknown sample or questioned item graphic source location based on the stable
from a crime scene or victim to a known sample isotope signature of an unknown specimen.
or exemplar taken from a subject or a subject’s These provenancing applications enable forensic
abode. In a first step, the samples are compared assignment of evidentiary material to potential
and a determination is made as to whether they geographic regions of origin by comparing the
appear to match. If they do, the second step is to isotopic composition of the sample to geospa-
determine the probability for the samples to have tial mapping of modeled stable isotope compo-
come from the same source. sitions of the same sample material based on
2. The second aim is to confirm or reject a hypothe- measured stable isotope signatures of authen-
sis about source or production method (synthetic tic samples [40–42]. Maps or “isoscapes” have
also been developed for the predicted isotopic
route) or geographic origin of an unknown sam-
composition of water throughout the world and
ple. This application requires knowledge of the
these maps serve as a principal basis for geo-
stable isotopic composition of authentic control
graphic provenancing of a wide range of mate-
samples, ideally in form of a database that can
rials such as plant, animal, and human tissue,
also provide information on background varia- even microbes, for which water is a precursor
tion in stable isotopic composition in relation substrate [28, 29, 43–47].
to source or origin. The stable isotope signature
of an unknown sample can then be compared
to a set of reference data and a determination Examples of Forensic Applications of Stable
can be made if its stable isotopic composition Isotope Signatures
matches the acceptance (or exclusion) criteria
as established from the collection of authentic Including investigations into breaches of laws and
observations. regulations that are not equally subject to criminal
 Stable Isotope Analysis: General Principles and Limitations 11

proceedings in all countries, a list of forensic appli- hair may be consistent with parts of Scandinavia,
cations of stable isotope signatures will consist of the parts of the former Soviet Union, parts of China,
following examples [8]: and parts of North America. The δ 13 C values from
the same hair sample indicate a large proportion of
• antidoping control [48–50]; the person’s dietary carbon intake comes from C4
• comparative analysis and provenancing of bacte- plants products (e.g., corn starch and corn syrup).
rial agents (anti-terrorism) [51–56]; From the four regions mentioned above only North
• comparative analysis and provenancing of coun- Americans are known to consume a lot of corn-
terfeit goods and money [57–60]; derived starch and sugar as part of their staple diet.
• comparative analysis and provenancing of explo- So, when looking at the δ 2 H values in conjunction
sives [8, 61–67]; with δ 13 C values from the same hair sample, this
• comparative analysis and source attribution of list of four regions can be narrowed down to just
packaging materials [65, 68, 69]; one. However, drawing the converse conclusion, i.e.,
• comparative analysis and provenancing of nar- using only human tissue δ 13 C values as a positive
cotic drugs [38, 41, 70–74]; predictor for North American provenance is not
• distinguishing between animals bred in captivity possible since most Africans consume a staple diet
and animals illegally caught or poached (wildlife rich in C4 plant products and so do people living
forensics) [43, 75]; in certain parts of South America (see Figure 3; for
• food adulteration, i.e., partial or complete sub- an in-depth discussion of the use of stable isotope
stitution of the authentic, natural, source or signatures for human provenancing, refer to the
origin-specific premium product with products of articles Stable Isotope Analysis: Bone and Teeth
different provenance, including synthetic prod- and Stable Isotope Analysis: Hair and Nail).
ucts [76–80]; In common with any other forensic analytical tech-
• human provenancing of living people (people nique involving several preparatory steps between
trafficking; antiterrorism) [47, 81–84]; sample collection and final analysis, each step will
• human provenancing of victims of serious crime make a contribution to the overall precision or repro-
or mass disasters [4, 5, 8, 85, 86]; ducibility of the final result. Similarly, the sequence
• source determination of environmental pollution from sample collection to final analysis via sample
(environmental forensics) [40, 87–92]. storage and sample preparation can also influence the
accuracy of the final result. However, while for tradi-
tional analytical techniques, loss of accuracy means
Caveats
either an error in quantification or an error in chem-
Limitations of Stable Isotope Analysis ical identification due to sample loss (poor recovery)
or artifact formation due to sample degradation or
Before discussing the underlying root causes that sample contamination, respectively, loss of accuracy
can impose limitations on the extent to which stable in SIA has a unique cause, mass discrimination lead-
isotope signatures can be used in a forensic context, ing to isotopic fractionation. In other words, the same
it is important to mention that the most appropriate process that enables us to distinguish between two
forensic use of stable isotope data for a given chemically identical drugs because they were derived
chemical element present in a sample is to apply from two different synthetic routes can become a pit-
them as a set of exclusion criteria but they should fall if sample preparation procedures do not result
not be seen or used as a predictor of a specific in a quantitative turnover or recovery of the sample.
sample characteristic such as geographic provenance. To avoid this pitfall, sample preparation procedures
While a select few exclusions to the latter rule employed in the process of SIA must be validated,
may exist, in general, stable isotope data should be demonstrating they are either free of isotopic frac-
looked at as descriptors that in their own right or tionation or for the isotopic fractionation to be quan-
in conjunction with other stable isotope data can tifiable and reproducible so its influence cannot be
provide a powerful set of exclusion criteria. For corrected for.
example, when trying to determine the recent life Another limitation relates to the nature of the
trajectory of an unidentified person, δ 2 H values from sample material itself. Chemicals that are the product
12 Stable Isotope Analysis: General Principles and Limitations

of well-controlled synthetic processes tend to be isotope abundances - a preliminary-study, Forensic Sci-

very homogeneous and both within sample isotopic ence International 71, 225–231.
variance and intra-batch isotopic variance are very [3] Casale, J., Casale, E., Collins, M., Morello, D.,
Cathapermal, S. & Panicker, S. (2006). Stable isotope
low. Naturally-made compounds are typically less analyses of heroin seized from the merchant vessel Pong
homogeneous so their natural or background isotopic Su, Journal of Forensic Sciences 51(3), 603–606.
variance can be so large to make it difficult to [4] Rauch, E., Rummel, S., Lehn, C. & Buettner, A. (2007).
distinguish between samples with different sample Origin assignment of unidentified corpses by use of
history. Sound knowledge, as in statistically sound stable isotope ratios of light (bio-) and heavy (geo-)
knowledge of background isotope variance or rather elements–a case report, Journal of Forensic Sciences
the lack thereof is hence a further limitation. To what 168, 215–218.
[5] Meier-Augenstein, W. & Fraser, I. (2008). Forensic
degree databases exist and how many entries they isotope analysis leads to identification of a mutilated
contain depends on several factors; (i) availability murder victim, Science & Justice 48(3), 153–159.
of control samples of known sample history and [6] Thomson, T.J.T. & Black, S.M. (2007). Forensic Human
provenance; (ii) quality of said control samples; and Identification, CRC Press, Boca Raton.
(iii) available funding of research in forensic sciences [7] Robert, D. & Blackledge, Ed. (2007). Forensic Analysis
to establish such databases. So, yet another limitation on the Cutting Edge, John Wiley & Sons, Inc., Hoboken.
can be the validity of the statistical approach taken [8] Meier-Augenstein, W. (2010). Stable Isotope Forensics:
An Introduction to the Forensic Application of Stable
to analyze stable isotope data and how to determine Isotope Analysis, John Wiley & Sons Ltd., Chichester.
the level of certainty or uncertainty of this technique [9] Fry, B. (2006). Stable Isotope Ecology, Springer, New
and its results. York.
Given the current drive for regulation and quality [10] NicDaeid, N. & Meier-Augenstein, W. (2008). Feasibil-
control in the provision of forensic science services, ity of source identification of seized street drug samples
limitations resulting from lack of funding for research by exploiting differences in isotopic composition at nat-
in forensic sciences and hence, lack of isotopic ural abundance level by GC/MS as compared to isotope
ratio mass spectrometry (IRMS), Forensic Science Inter-
databases will hopefully soon be a thing of the past national 174(2–3), 259–261.
since one cannot have one without the other. While [11] Sharp, Z.D. (2007). Principles of Stable Isotope Geo-
the number of cases where stable isotope data have chemistry, Pearson Prentice Hall, Upper Saddle River.
been given in evidence in a court of law are few [12] Meier-Augenstein, W., Watt, P.W. & Langhans, C.D.
and far between, given the increase in cases relying (1996). Influence of gas-chromatographic parameters
on stable isotope signatures or profiles as sources of on measurement of c-13/c-12 isotope ratios by gas-
intelligence to inform, facilitate, and focus criminal liquid-chromatography combustion isotope ratio mass-
spectrometry. 1, Journal of Chromatography A 752,
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stable isotope data in furtherance of their case. The module for internal isotopic calibration in high preci-
anticipation of this happening has already resulted in sion gas chromatography combustion-isotope ratio mass
the publication of an article addressing the rules of spectrometry, Rapid Communications in Mass Spectrom-
admissibility of stable isotope data in a court of law etry 11, 1775–1780.
[14] Werner, R.A. & Brand, W.A. (2001). Referencing strate-
from a US perspective [39].
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Rapid Communications in Mass Spectrometry 15(7),
501–519.
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