The heart of forensic science is application of the scientific method and analytical approaches to answer questions central to solving a crime: Who, What, When, Where, and How. Forensic practitioners use fundamentals of chemistry and physics to examine evidence and infer its origin. In this regard, ecological researchers have had a significant impact on forensic science through the development and application of a specialized measurement technique—isotope analysis—for examining evidence. Here, we review the utility of isotope analysis in forensic settings from an ecological perspective, concentrating on work from the Americas completed within the last three decades. Our primary focus is on combining plant and animal physiological models with isotope analyses for source inference. Examples of the forensic application of isotopes—including stable isotopes, radiogenic isotopes, and radioisotopes—span from cotton used in counterfeit bills to anthrax shipped through the U.S. Postal Service and from beer adulterated with cheap adjuncts to human remains discovered in shallow graves. Recent methodological developments and the generation of isotope landscapes, or isoscapes, for data interpretation promise that isotope analysis will be a useful tool in ecological and forensic studies for decades to come.

Isotope ratio analysis has been shown to discriminate samples of forensic interest and to link many synthesized and natural materials to their precursors when traditional chemical and physical analyses cannot. Successful application of stable isotope analysis to chemicals of interest requires a background of likely variations in stable isotope ratios; often, this background population can be generated from analysis of possible precursors and the relationships of stable isotopes of precursor(s) to product(s), which may depend on synthesis techniques. Here we measured the carbon (13C/12C) and hydrogen (2H/1H) isotope ratios of the oft-illicitly manufactured explosive triacetone triperoxide, TATP, and one of its precursors, acetone. As acetone is the sole source of carbon and hydrogen to TATP, a survey of acetone from 12 countries was conducted to explore the breadth of 13C/12C and 2H/1H variation in the precursor, and therefore, its product. Carbon and hydrogen isotope ratios were measured using continuous flow isotope ratio mass spectrometry (IRMS) techniques. We observed greater ranges in both C and H isotope ratios of acetone than previously published; we also found that country-of-purchase was a large contributing factor to the observed variation, larger than acetone grade and brand. Following clandestine production methods, we observed that the stable isotope ratios of TATP retained the stable isotope signatures of acetone used in synthesis. We confirmed the robustness of TATP carbon isotope ratios to both recrystallization and time-dependent sublimation, important considerations when faced with the task of practical sampling of potential unexploded TATP from a crime scene.

Isotope ratio mass spectrometry (IRMS) techniques have been used for decades to characterize explosives alongside traditional chemical analyses. We present here a broadly applicable framework for methodologically applying a variety of separation techniques to explosive mixtures and preparing different explosive components for stable isotope analysis. This framework allows an analyst to collect the most characteristic information possible from an explosive sample, by analyzing multiple components with independent isotope signatures to produce multivariate datasets for discrimination. A case illustration demonstrating the application of methods in the framework to plastic explosives (specifically C-4 and Semtex) has been prepared separately. In developing this framework, we focused on explosives samples that may contain RDX, HMX, PETN, TNT, AN, and/or nitrocellulose along with various binders, plasticizers, oils, and other additives. This paper describes the theory and processes used to develop a component-specific approach to prepare explosives samples for isotope ratio analysis, focusing specifically on optimization of solvent extraction methods. Other methods used in framework development include gravimetric analysis and HPLC.

An improved method for the separation of the components of plastic explosives is described with the goal of stable isotope ratio analysis of each isolated component. The binder and plasticizer of a plastic explosive were extracted into cyclohexane, leaving the explosive components and other insoluble material. The plasticizers, oils, and additives were next extracted from the dried cyclohexane-soluble fraction into acetone, leaving the polymeric binder. Plasticizers, oils, and additives were identified by GC–MS and individual plasticizers, oils, dyes, and antioxidants were isolated by GC and/or flash chromatography free of interfering materials. The explosive components were extracted from the cyclohexane-insoluble fraction with acetone, and then quantitatively analyzed for individual explosive compounds via HPLC-UV/Vis spectroscopy. Individual explosive compounds were isolated via preparative HPLC. The separation process was validated by gravimetric, HPLC-UV/Vis, EA, GC–MS, TC/EA, and FTIR results consistent with the expected formulation of the plastic explosives. The isolated components were analyzed for component-specific stable isotope ratios. Control mixtures of previously characterized components of two common plastic explosive formulations were also separated and analyzed. These isolated components retained the isotope ratios prior to mixture, demonstrating the robustness and reliability of the technique. Two example C-4 explosives from different sources, with indistinguishable chemical composition and raw explosive 13C/12C and 15N/14N isotope ratios, were extracted and analyzed. The resultant binder and plasticizer 2H/1H and 13C/12C results were used to discriminate between the two C-4 samples that were otherwise apparently identical.

RATIONALE Elemental analyzer systems generate N(2) and CO(2) for elemental composition and isotope ratio measurements. As quantitative conversion of nitrogen in some materials (i.e., nitrate salts and nitro-organic compounds) is difficult, this study tests a recently published method - thermal decomposition without the addition of O(2) - for the analysis of these materials. METHODS Elemental analyzer/isotope ratio mass spectrometry (EA/IRMS) was used to compare the traditional combustion method (CM) and the thermal decomposition method (TDM), where additional O(2) is eliminated from the reaction. The comparisons used organic and inorganic materials with oxidized and/or reduced nitrogen and included ureas, nitrate salts, ammonium sulfate, nitro esters, and nitramines. Previous TDM applications were limited to nitrate salts and ammonium sulfate. The measurement precision and accuracy were compared to determine the effectiveness of converting materials containing different fractions of oxidized nitrogen into N(2). RESULTS The δ(13) C(VPDB) values were not meaningfully different when measured via CM or TDM, allowing for the analysis of multiple elements in one sample. For materials containing oxidized nitrogen, (15) N measurements made using thermal decomposition were more precise than those made using combustion. The precision was similar between the methods for materials containing reduced nitrogen. The %N values were closer to theoretical when measured by TDM than by CM. The δ(15) N(AIR) values of purchased nitrate salts and ureas were nearer to the known values when analyzed using thermal decomposition than using combustion. CONCLUSIONS The thermal decomposition method addresses insufficient recovery of nitrogen during elemental analysis in a variety of organic and inorganic materials. Its implementation requires relatively few changes to the elemental analyzer. Using TDM, it is possible to directly calibrate certain organic materials to international nitrate isotope reference materials without off-line preparation.

RATIONALE Because of the threat of bombings using improvised explosives containing ammonium nitrate (AN), law enforcement and intelligence communities have been interested in stable isotope techniques for tracking and discriminating AN sources. Separate analysis of the AN component ions ammonium and nitrate would add discriminatory power to these techniques. METHODS Ammonium ions in dissolved AN solution were isolated from samples by precipitation using sodium tetraphenylborate solution. We tested the isolation of ammonium from nitrates using solutions of ammonium and nitrate salts with different (15)N/(14)N isotope ratios. Ammonium tetraphenylborate and AN were separately analyzed for their (15)N/(14)N isotope ratios using EA-ConFlo-IRMS, and the (15)N/(14)N isotope ratios of the nitrate ions were calculated using mass balance. Ammonium and nitrate nitrogen isotope ratios were plotted as two separate variables. RESULTS Isolation of ammonium precipitate from solutions containing dissolved nitrates did not influence the nitrogen isotope ratios of test ammonium salts. A survey set of 42 AN samples showed that the ammonium and nitrate (15)N/(14)N isotope ratios were not significantly correlated, and the paired mean differences were not statistically significant. Both ammonium and nitrate were depleted in (15)N relative to their theoretical atmospheric sources. CONCLUSIONS Isolation of the ammonium ion from AN adds another dimension for the discrimination of forensic AN samples. This technique using sodium tetraphenylborate is robust and does not require specialized equipment. Our observations indicated that ammonium nitrogen and nitrate nitrogen have independent sources of isotopic variation.

RDX and HMX are explosive compounds commonly used by the military and also occasionally associated with acts of terrorism. The isotopic characterization of an explosive can be a powerful approach to link evidence to an event or an explosives cache. We sampled explosive products and their reactants from commercial RDX manufacturers that used the direct nitration and/or the Bachmann synthesis process, and then analyzed these materials for carbon and nitrogen isotope ratios. For manufacturers using the Bachmann process, RDX 13C enrichment relative to the hexamine substrate was small (+0.9‰) compared to RDX produced using the direct nitration process (+8.2‰ to +12.0‰). RDX 15N depletion relative to the nitrogen-containing substrates (−3.6‰) was smaller in the Bachmann process than in the direct nitration process (−12.6‰ to −10.6‰). The sign and scale of these differences agree with theorized mechanisms of mass-dependent fractionation. We also examined the isotopic relationship between RDX and HMX isolated from explosive samples. The δ13C and δ15N values of RDX generally matched those of the HMX with few exceptions, most notably from a manufacturer known to make RDX using two different synthesis processes. The range in δ13C values of RDX in a survey of 100 samples from 12 manufacturers spanned 33‰ while the range spanned by δ15N values was 26‰; these ranges were much greater than any previously published observations. Understanding the relationship between products and reactants further explains the observed variation in industrially manufactured RDX and can be used as a diagnostic tool to analyze explosives found at a crime scene.

Isotope ratio analysis allows forensic investigators to discriminate materials that are chemically identical but differ in their isotope ratios. Here we focused on the discrimination of pentaerythritol tetranitrate (PETN), an explosive with military and civilian applications, using carbon (δ13C) and nitrogen (δ15N) isotope ratios. Our goal was to understand some of the factors influencing the isotope ratios of commercially manufactured PETN. PETN was isolated from bulk explosives using preparative HPLC, which reduced chemical and isotopic within-sample variability. We observed isotope ratio variation in a survey of 175 PETN samples from 22 manufacturing facilities, with δ13C values ranging from −49.7‰ to −28.0‰ and δ15N values ranging from −48.6‰ to +6.2‰. Both within-sample variability and variation of PETN within an explosive block were much smaller than between-sample variations. Isotopic ratios of PETN were shown to discriminate explosive blocks from the same manufacturer, whereas explosive component composition measurements by HPLC were not able to do so. Using samples collected from three industrial PETN manufacturers, we investigated the isotopic relationship between PETN and its reactants, pentaerythritol (PE) and nitric acid. Our observations showed that δ13C values of PETN were indistinguishable from that of the reactant pentaerythritol. Isotopic separation between nitric acid and PETN was consistent within each sampled manufacturer but differed among manufacturers, and was likely dependent upon reaction conditions. These data indicate that δ13C variation in PETN is dependent on δ13C variation of PE supplies, while δ15N variation in PETN is due to both nitric acid δ15N and reaction conditions.

Metabolic processes result in the release and exchange of H and O atoms from organic material as well as some inorganic salts and gases. These fluxes of H and O atoms into intracellular water result in an isotopic gradient that can be measured experimentally. Using isotope ratio mass spectroscopy, we revealed that slightly over 50% of the H and O atoms in the intracellular water of exponentially-growing cultured Rat-1 fibroblasts were isotopically distinct from growth medium water. We then employed infrared spectromicroscopy to detect in real time the flux of H atoms in these same cells. Importantly, both of these techniques indicate that the H and O fluxes are dependent on metabolic processes; cells that are in lag phase or are quiescent exhibit a much smaller flux. In addition, water extracted from the muscle tissue of rats contained a population of H and O atoms that were isotopically distinct from body water, consistent with the results obtained using the cultured Rat-1 fibroblasts. Together these data demonstrate that metabolic processes produce fluxes of H and O atoms into intracellular water, and that these fluxes can be detected and measured in both cultured mammalian cells and in mammalian tissue.

Hydrogen peroxide (H(2)O(2)) is a widely used oxidizer with many commercial applications; unfortunately, it also has terrorist-related uses. We analyzed 97 hydrogen peroxide solutions representing four grades purchased across the United States and in Mexico. As expected, the range of hydrogen (δ(2)H, 230‰) and oxygen (δ(18)O, 24‰) isotope values of the H(2)O(2) solutions was large, reflecting the broad isotopic range of dilution waters. This resulted in predictable linear relationships of δ(2)H and δ(18)O values of H(2)O(2) solutions that were near parallel to the Meteoric Water Line (MWL), offset by the concentration of H(2)O(2) in the solution. By grade, dilute (3 to 35%) H(2)O(2) solutions were not statistically different in slope. Although the δ(2)H values of manufactured H(2)O(2) could be different from those of water, rapid H(2)O(2)-H(2)O exchange of H atoms eliminated any distinct isotope signal. We developed a method to measure the δ(18)O value of H(2)O(2) independent of dilution water by directly measuring O(2) gas generated from a catalase-induced disproportionation reaction. We predicted that the δ(18)O values of H(2)O(2) would be similar to that of atmospheric oxygen (+23.5‰), the predominant source of oxygen in the most common H(2)O(2) manufacturing process (median disproportionated δ(18)O=23.8‰). The predictable H-O relationships in H(2)O(2) solutions make it possible to distinguish commercial dilutions from clandestine concentration practices. Future applications of this work include synthesis studies that investigate the chemical link between H(2)O(2) reagents and peroxide-based explosive products, which may assist law enforcement in criminal investigations.

Measurements were made on the natural abundance 14C content (Δ14C) of cocaine specimens seized between 2003 and 2009. The objective of this study was to determine the extent to which Δ14C analyses could quantify the “age” of recent cocaine seizures. Here “age” of a seized cocaine specimen is defined as the time period between when a coca leaf was harvested in South America and its seizure as cocaine at either the international or domestic street levels. Based on Δ14C analyses of seizure specimens, there were no statistically significant differences in the ages of domestic cocaine HCl and cocaine base specimens seized on the streets in different locations across the United States. Between 2007 and 2009, the average age of a street-level cocaine seizure in the United States was 24.6±1.1months. Cocaine shipment seizures that were in excess of 150kg during this time period had an average age of 18.2±1.4months, whereas smaller shipment seizures were significantly older with an average age of 22.3±0.6months. Analyses of the largest cocaine shipment seizures suggested that these seizures were composed of specimens with different ages, possibly representing accumulations over as much as a 31-month period.

Two Δ14C calibration curves have been produced that allow determination of the statistical average age of coca leaf and cocaine base specimens produced for the time period 1979–2009. These calibration curves are based on field collections of specimens in Bolivia, Colombia, Ecuador, and Peru. The coca leaf F14C and Δ14C calibration curves can be used to predict the ages of botanical tissues collected in tropical South America and possibly extended to other tropical locations. The cocaine F14C and Δ14C calibration curves can be used to predict the ages of seized cocaine specimens. Because the Δ14C of the atmosphere is diminishing, the precision of this approach for age determinations will continue to get less precise over time as atmospheric 14C content continues to decline.

This chapter tests the premise that a single sample from an upper trophic level pelagic predator fish could provide information on both the trophic level of the fish and the δ15N value at the base of the food web. This chapter analyzes the nitrogen isotopic composition of individual amino acids in white muscle tissue (WMT) of yellowfin tuna (Thunnus albacares) from the eastern tropical Pacific (ETP) to determine if the observed variations in the δ15N values of WMT are primarily controlled by the nitrogen isotopic composition at the base of the food web or the trophic level of the tuna. The difference between the δ15N values of source and trophic amino acids is used to estimate the trophic level of yellowfin, and this is compared to the estimates based on two independent methods, stomach content analysis and the isotopic difference between the δ15N values of mesozooplankton and yellowfin tuna in the ETP. It is found that nitrogen isotopic analyses of individual amino acids in tuna can be used to distinguish between nutrient and trophic dynamics in pelagic ecosystems, and the implications of these results are discussed for investigating the long-term impact of commercial fishing on the food web structure of marine ecosystems.

It is generally believed that water transport across biological membranes is essentially a near-instantaneous process, with water molecules diffusing directly across the membrane as well as through pores such as aquaporins. As a result of these processes by which water can equilibrate across a membrane, a common assumption is that intracellular water is isotopically indistinguishable from extracellular water. To test this assumption directly, we measured the hydrogen isotope ratio of intracellular water in Escherichia coli cells. Our results demonstrate that more than 50% of the intracellular water hydrogen atoms in log-phase E. coli cells are isotopically distinct from the growth medium water and that these isotopically distinct hydrogen atoms are derived from metabolic processes. As expected, the (2)H/(1)H isotope ratio of intracellular water from log-phase cells showed an appreciably larger contribution from metabolic water than did intracellular water from stationary-phase cells (53 +/- 12 and 23 +/- 5%, respectively). The (2)H/(1)H isotope ratio of intracellular water was also monitored indirectly by measuring the isotope ratio of fatty acids, metabolites that are known to incorporate hydrogen atoms from water during biosynthesis. Significantly, the difference in the isotopic composition of intracellular water from log- to stationary-phase E. coli cells was reflected in the hydrogen isotope ratio of individual fatty acids harvested at the two different times, indicating that the isotope ratio of metabolites can be used as an indirect probe of metabolic activity. Together, these results demonstrate that contrary to the common assumption that intracellular water is isotopically identical to extracellular water, these two pools of water can actually be quite distinct.