Michael J. Lott

Tracing the geographical origin of cocaine

Here we show that cocaine originating from different geographic regions in South America can be identified by its isotope-ratio signature. The distinct carbon (δ13C) and nitrogen (δ15N) isotope-ratio combinations allow the country of origin to be determined for the principal coca-growing regions along the Andean Ridge. By combining this information with detectable differences in the patterns of the trace alkaloids truxilline and trimethoxycocaine, we correctly identified the source of 96% of 200 cocaine samples.

Geo-location of heroin and cocaine by stable isotope ratios

Analyses of the carbon and nitrogen stable isotope ratios in heroin and cocaine samples obtained from different geographic regions indicated stable isotope ratio combinations that were strongly correlated with geographic location. Further analyses of the isotope ratios of morphine derived from the deacetylation of heroin exhibited more pronounced isotopic differences among regions, increasing its potential as a tool for geo-location and for sample-to-sample comparison.

Microbe forensics: Oxygen and hydrogen stable isotope ratios in Bacillus subtilis cells and spores

Bacillus subtilis, a Gram-positive, endospore-forming soil bacterium, was grown in media made with water of varying oxygen (δ18O) and hydrogen (δD) stable isotope ratios. Logarithmically growing cells and spores were each harvested from the cultures and their δ18O and δD values determined. Oxygen and hydrogen stable isotope ratios of organic matter were linearly related with those of the media water. We used the relationships determined in these experiments to calculate the effective whole-cell fractionation factors between water and organic matter for B. subtilis. We then predicted the δ18O and δD values of spores produced in nutritionally identical media and local water sources for five different locations around the United States. Each of the measured δ18O and δD values of the spores matched the predicted values within a 95% confidence interval, indicating that stable isotope ratio analyses may be a powerful tool for tracing the geographic point-of-origin for microbial products.

Isotopic fractionation of carbon and nitrogen during the illicit processing of cocaine and heroin in South America.

The forensic application of stable isotope analysis to cocaine and heroin for geolocation of exhibits must take into account the possible enrichment and/or depletion of 13C and 15N during the illicit manufacturing process. Continuous-flow elemental analysis-isotope ratio mass spectrometry was utilized to measure changes in the stable isotope ratios of carbon and nitrogen for both cocaine (N = 92) and heroin/morphine (N = 81) exhibits derived from illicit manufacturing processes utilized by South American clandestine chemists. In controlled settings in South America, there was no siginficiant carbon isotope fractionation during the conversion of cocaine base to cocaine HCI using current illict methodologies. In contrast, nitrogen isotope fractionation for this conversion was 1 per thousand. There was a kinetic carbon isotope ratio fractionation during the acetylation of Colombian morphine to heroin and as a result heroin exhibits will almost always have more negative delta13C values than the original morphine. There was an isotopic fractionation against 15N during the acetylation of morphine base to heroin base, but this effect was not expressed since all of the heroin base was precipitated during the manufacturing process. However, the clandestine process of converting a single batch of heroin base usually involved two consecutive crops of heroin HCl and the latter crop was isotopically depleted as expected from a Rayleigh distillation process. When heroin was deacetylated to morphine, the morphine produced resulted in delta13C values that were indistinguishable from the original morphine. The kinetic carbon isotope fractionation factor for the South American process of morphine acetylation was -1.8 per thousand, allowing calculation of the delta13C values of the acetic anhydride from deacetylated heroin delta13C values.

Stable Isotope Ratios as a Tool in Microbial Forensics—Part 1. Microbial Isotopic Composition as a Function of Growth Medium ∗

The stable isotope ratios of a seized pathogen culture could potentially reveal information about the environment in which the agent was produced. In this paper we describe general relationships between stable isotopes of carbon, nitrogen, and hydrogen in bacteriological culture media and spores of Bacillus subtilis, an endospore-forming soil bacterium. In numerous media that varied both in nutrient composition and water stable isotope ratios, medium to spore enrichment in carbon isotopes was 0.3 +/- 2.0% per thousand (parts per thousand), and in nitrogen, 4.5 +/- 0.7% per thousand. We achieved mass balance for the contribution of hydrogen isotopes from nutrients (70%) and water (30%) to spores in independent experiments by varying the isotope ratios of nutrients or water. A model was derived for predicting the isotope ratio values of spores from those in nutrients and water.

Stable Isotope Ratios as a Tool in Microbial Forensics—Part 2. Isotopic Variation Among Different Growth Media as a Tool for Sourcing Origins of Bacterial Cells or Spores ∗

Since the anthrax attacks of 2001 the need for methods to trace the origins of microbial agents has become urgent. The stable isotope ratios of bacteria record information from both the nutrients and the water used to make their culture media and could potentially be used to provide information about their growth environment. We present a survey of carbon (C), nitrogen (N), and hydrogen (H) stable isotope ratios in 516 samples of bacteriological culture media. The observed variation was consistent with expected isotopic variation in the plant and animal products upon which the media are based. The variation is sufficient to translate into substantial isotope variation in cultures grown on different batches of media, and thus to allow investigators to determine whether seized media could have been used to produce seized bioweapons agents.

Carbon and nitrogen isotope ratios of factory-produced RDX and HMX

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 (13)C 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 (15)N 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 δ(13)C and δ(15)N 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 δ(13)C values of RDX in a survey of 100 samples from 12 manufacturers spanned 33‰ while the range spanned by δ(15)N 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.

Detection of Metabolic Fluxes of O and H Atoms into Intracellular Water in Mammalian Cells

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.

Stable isotope ratios as a tool in microbial forensics--part 3. Effect of culturing on agar-containing growth media.

Stable isotope ratios of hydrogen and oxygen in microbes have been shown to be functions of the corresponding isotope ratios of the water with which the culture medium was prepared, and thus to contain a potential geographic signal. Water can evaporate from agar (solid) media during culturing, changing its isotope ratios. Here we describe the effect of drying on the isotope ratios of water extracted from agar media and the H and O stable isotope ratios ratios of Bacillus subtilis spores cultured on agar. The delta2H vs delta18O relationship of water in Petri dish agar was surprisingly constant during evaporation regardless of the ambient relative humidity, making it possible to calculate the approximate isotope ratios of the original water, even in significantly evaporated agar. The H stable isotope ratios of spores cultured on agar remained relatively unchanged as the agar dried, but the O ratio became significantly enriched.

Hydrogen and oxygen isotope values in hydrogen peroxide

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.