Isotope Ratio Mass Spectrometry
Isotope ratio mass spectrometry (IRMS) is an analytical technique that allows for the measurement of variations in isotopic abundances of certain light elements, namely hydrogen, carbon, nitrogen, and oxygen.
Isotopes are atoms of an element that differ only in the number of neutrons present in their nuclei, thus having different mass numbers. For instance, carbon has three isotopes that are present in nature; 12C, 13C and 14C. The light isotope, 12C, is the dominant isotope, with a natural abundance of just under 99%. Variations in the isotopic composition of substances can arise through various chemical, biological and physical processes (primarily kinetic and thermodynamic isotope effects) which bring about what is known as isotopic fractionation. Principally it is the lighter elements that are affected by isotopic fractionation (Benson et al, 2006). The isotope ratio values caused by different fractionation effects are often unique to certain origins, manufacturing processes, purities and so on. It is these minute differences that are determined and used in forensic IRMS to draw connections, distinguish between, and even trace samples.
Initially samples will be separated and introduced into the IRMS by an additional technique such as a gas chromatograph (GC). In this instance, the injected sample will be vaporised and carried by an inert carrier gas onto the analytical column, where the sample components will be separated based on their varying interactions with the carrier gas and the stationary phase within the column. Compounds will then elute out of the column into the next section of the instrument, where the IRMS process continues. Samples may be introduced into the mass spectrometer as a pure gas by means of combustion.
GC-C-IRMS (gas chromatography-combustion-isotope ratio mass spectrometry) systems typically involve the use of one or more reactors – a combustion reactor and, in some cases, a reduction reactor. The temperature at which the combustion reactor is held can vary depending on the application, but is typically in the region of 1000oC. These reactors can also vary in composition, though will often contain a mixture of copper oxide and nickel oxide. At this point the separated sample will be combusted into a simple gas that is representative of the original analyte, such as CO2, N2, or H2. Following this, the removal of excess oxygen (eg from nitrogen oxide into N2) can be carried out in reduction reactor, generally at a slightly lower temperature. Water can be removed using a substance known as Nafion, after which the sample is passed into the mass spectrometer.
In the ion source of the mass spectrometer, gas molecules are ionised by a beam of electrons in an evacuated chamber. The newly formed ions are then focussed and accelerated, the magnetic field determining the trajectory of the ions and thus separating them. Ions of with different m/z ratios will be distinguished from one another and can then be quantified. For instance, when measuring the isotopic difference of carbon atoms, mass-to-charge (m/z) ratios of 44, 45 and 46 will be monitored, each captured by individual Faraday cups, in order to account for CO2 composed of the three isotopes of carbon; 12C, 13C and 14C. Each ion reaching a cup will contribute charge, which will them be amplified. The abundance of isotopes are then typically compared with the isotope ratios of specific standards.
After analysis, the variations in stable isotope abundances are expressed as a ratio, known as the delta notation, calculated by relating the ratio of heavy to light isotopes in the sample to those in a known standard reference material. Substances analysed will typically have a negative delta value due to being depleted in the heavier isotope relative to a reference standard. Various forms of IRMS exist, with numerous analytical techniques combined with it to achieve different results.
IRMS can be applied to a wide range of areas of forensic science. The ability to distinguish between, or conversely link, seemingly identical samples can be pivotal in a forensic investigation. For instance, using IRMS it could be possible to establish whether or not two different samples of heroin likely originated from the same source. Isotopic fractionation can occur as the result of different growing or synthesis conditions and later how substances were handled and stored, which can result in different batches of what is chemically the same substances having different isotopic compositions.
A particularly interesting use of stable isotope analysis in forensic science is for the purpose of tracing individuals to certain locations. The atoms making up a person’s body are largely fed by the food and drink consumed by that person, atoms which will consist of varying amounts of isotopes. The composition of water (and thus the drinking water we consume) is affected by various isotopic fractionation effects, including precipitation amount, temperature, altitude and seasonality. The combination of these various effects, and others, result in water developing an isotopic composition specific to a particular region. It is not only possible to link an individual to a certain area based on this evidence, but also trace their movement from one location to another over a time period. Certain body tissues, such as hair and fingernails, can be subjected to stable isotope analysis to provide a kind of isotopic timeline due to the linear fashion in which they record isotopic compositions. Just as oxygen isotopic ratios can be indicative of the water consumed by a person, other elements such as carbon and nitrogen can be studied to examine the food eaten. Nitrogen isotope levels often differ between omnivores, vegetarians and vegans, and carbon isotopes exist in different concentrations in certain food products depending on the type of photosynthesis used by that plant. Thus the study of these isotopes can provide clues as to a person’s diet. Information obtained regarding a person’s lifestyle (eg. diet) and their location and migration patterns may prove beneficial when attempting to identify human remains, particularly if the more typically utilised dental records and DNA are not useful.
Stable isotope analysis has been utilised in wildlife forensics, particularly with regards to illegal poaching. Elephant bone and ivory have been subjected to IRMS analysis (Stelling et al, 2001) in hopes of distinguishing between different populations of protected animals. The ability to do so would enable suspected illegal ivory to be traced to particular regions, thus aiding in punishing and preventing poachers who kill elephants for their ivory.
The list of applications goes on, including pharmaceutical counterfeit operations using IRMS to distinguish between genuine drug products and counterfeits, environmental investigations utilising the technique to identify and source a pollutant in the environment, and distinguishing between inks. The potential use of IRMS in the forensics sciences is vast, with the applications covered here representing only a handful of cases. Although the complexity and expense of this analytical technique renders it far from being a commonplace method, the use of isotope ratio mass spectrometry in the field of forensics is increasing.
Benson et al., 2006 S. Benson, C. Lennard, P. Maynard, C. Roux Forensic applications of isotope ratio mass spectrometry — a review. Forensic Science International, 157 (2006), pp. 1–22
Carter J. F, and Barwick V. J. (Eds), Good practice guide for isotope ratio mass spectrometry, FIRMS (2011)
Chesson, L. A, Tipple, B. J, Barnette, J. E, Cerling, T. E, Ehleringer, J. R. The potential for application of ink stable isotope analysis in questioned document examination. Science & Justice (2014).
Marimon, R. M. Perona, J. Teixidor, P. Isotope Ratio Mass Spectrometry. Handbook of Instrumental Techniques from CCiTUB (2012).
M.A. Stelling, G.J.Q. Van der Peijl, Forensic methods for criminal investigations related to endangered species. Netherlands Forensic Institute report, December 2001.