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.     

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 hydrogen isotope analysis as a method to identify illegally trapped songbirds

We measured stable hydrogen isotope ratios in the primary feathers of two subspecies of goldfinches, confiscated by the Police and the Royal Society for the Prevention of Cruelty to Animals (RSPCA) on suspicion that they had been illegally taken from the wild. We found significant differences in the delta2H values of the two subspecies indicating that they were sourced from different geographical regions. Our results correlated with isotopic precipitation maps and with the known distribution of the two subspecies of goldfinch. We believe that this technique could be used by law enforcement agencies to determine the origin of birds in cases where the species or subspecies involved are geographically distinct.     

Isotope ratio mass spectrometry as a tool for forensic investigation (examples from recent studies).

The versatility of isotope ratio mass spectrometry is demonstrated by reference to diverse case studies. Variations in the natural isotopic composition of non-biological, organic materials are compared as a means by which samples may be associated or discriminated. These techniques may be used to augment or compliment conventional forensic methodologies. delta13C analysis was used to demonstrate that different masking tape had been recovered in two, apparently similar cases, involving the smuggling of money. Visually similar ecstasy tablets were compared by consideration of the delta13C and delta15N composition of MDMA extracted from the tablets. Although only a limited number of tablets were analysed, the isotopic similarity between two different seizures was sufficient to induce a guilty plea from a person suspected of possessing both. A combination of delta2H, delta13C, delta15N and delta18O together with GC-MS analyses were applied to small samples of seized heroin. Although GC-MS analysis indicated differences between the chemical composition of two of the heroin samples, isotopic analysis suggested similarities, which were confirmed by further delta2H, delta13C and delta18O isotopic analysis of the clingfilm in which the samples were wrapped.     

Origin assignment of unidentified corpses by use of stable isotope ratios of light (bio-) and heavy (geo-) elements--a case report

An unknown male body was found near an expressway in Germany. As different criminalistic and forensic methods (e.g. tooth status, fingerprint or DNA-analysis) could not help to identify the person, multielement stable isotope investigations were applied. The combined analysis of stable isotope ratios of light (H, C, N) and heavy elements (Pb, Sr) on the man's body tissues supported to assign him to Romania. The case report demonstrates an application of multielement-isotope analysis in the forensic fields and its potential.     

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.     

Assessing Carbon and Hydrogen Isotopic Fractionation of Diesel Fuel n-alkanes During Progressive Evaporation

Compound-specific isotope analysis offers potential for fingerprinting of diesel fuels, however, possible confounding effects of isotopic fractionation due to evaporation need to be assessed. This study measured the fractionation of the stable carbon and hydrogen isotopes in n-alkane compounds in neat diesel fuel during evaporation. Isotope ratios were measured using a continuous flow gas chromatograph/isotope ratio mass spectrometer. Diesel samples were progressively evaporated at 24 ± 2°C for 21 days. Increasing depletion of deuterium in nC₁₂–nC₁₇ alkanes in the remaining liquid with increasing carbon chain length was observed. Negligible carbon isotope fractionation was observed. Preferential vaporization was measured for the shorter chain n-alkanes and the trend decreased with increasing chain length. The decrease in δ²H values indicates the preferential vaporization of the isotopically heavier species consistent with available quantitative data for hydrocarbons. These results are most important in the application of stable isotope technology to forensic analysis of diesel.     

Stable isotope analysis of human hair and nail samples: the effects of storage on samples

When submitting samples for analysis, maintaining sample integrity is essential. Appropriate packaging must be used to prevent damage, contamination or loss of sample. This is particularly important for stable isotope analysis by isotope ratio mass spectrometry as this technique is capable of detecting subtle differences in isotopic composition with great precision. In a novel study, scalp hair and fingernail samples were placed in five different types of packaging, routinely used in forensic laboratories and stored for 6 weeks and 6 months. Samples were subsequently cleaned and submitted for (13)C/(12)C, (15)N/(14)N, (2)H/(1)H and (18)O/(16)O analysis. Results from (13)C analysis indicate that type of packaging can cause slight changes in (13)C abundance over time. Differences were noted in the (15)N isotope signatures of both hair and nail samples after 6-week storage, but not after 6 months. This apparent discrepancy could be a result of the packaging not being properly sealed in the 6 weeks study. Fewer differences were noted when analyzing samples for (2)H and (18)O abundance.     

Stable isotope models to predict geographic origin and cultivation conditions of marijuana

Here we describe stable isotope based models using hydrogen and carbon isotope ratios to predict geographic region-of-origin and growth environment for marijuana, with the intent of applying these models to analyses of marijuana trafficking in the USA. The models were developed on the basis of eradication specimens and border specimens seized throughout the USA. We tested reliability of the geographic region-of-origin and growth environment models with a “blind” set of 60 marijuana eradication specimens obtained from counties throughout the USA. The two geographic region-of-origin model predictions were 60–67% reliable and cultivation environment model predictions were 86% accurate for the blind specimens. We demonstrate here that stable isotope ratio analysis of marijuana seizures can significantly improve our understanding of marijuana distribution networks and it is for that purpose that these models were developed.     

Marine bacteria comprise a possible indicator of drowning in seawater

To investigate the effectiveness of marine bacteria as a new marker of drowning in seawater, we determined the optimal conditions of media required to selectively detect marine bacteria and applied the technique to drowned cadavers. We incubated model blood samples (n=20 per group) mixed with seawater, river, tap or muddy water on agar plates (Todd Hewitt, TH; Marine 2216, M2216) and determined the NaCl concentration required to selectively detect marine bacteria. We also used TCBS agar plates without manipulation to isolate Vibrio spp. Among the culture media, TH agar was superior. Bioluminescent colonies were detected only in blood mixed with seawater. Blue colonies stained using the cytochrome oxidase test (COT), were detected in blood mixed with both sea and river water. However when the NaCl concentration was above 4%, COT stained colonies were detectable only in blood mixed with seawater. We subsequently used 2, 3 and 4% NaCl in TH and TCBS agar to examine blood from victims who had drowned in seawater (n=8) and in fresh water (n=7), as well as from victims who died near aquatic environments (non drowned; dry-land control, n=7). Bioluminescent colonies were detectable on 2-4% NaCl TH agar only from two victims that drowned in seawater. Bioluminescent colonies did not grow on TCBS agar. Blue colonies from all cadavers that had drowned in seawater (8/8) and in four of those that had drowned in fresh water (4/7) proliferated on TH agar containing 2% and/or 3% NaCl, but at 4% NaCl such colonies were detected only from cadavers that had drowned in seawater (8/8). Colonies from only one cadaver from seawater grew on TCBS agar. Furthermore, neither bioluminescent nor blue colonies were detected on TH agar containing 4% NaCl in samples from two cadavers found in an estuary (brackish water) who were thought to have been carried from areas of fresh water. Homologous analyses of the 16S rRNA gene revealed that the dominant colonies on TH agar containing 4% NaCl were marine bacteria (Photobacterium, Vibrio, Shewanella, Psychrobacter). Thus, proliferating bioluminescent and/or blue colonies detected in the blood of immersed cadavers using 4% NaCl TH agar, could help to establish drowning in seawater.     

Traveling There and Back Again: A Fingernail's Tale

This study focuses on the examination of stable isotope biomarkers in human fingernails of travelers who ventured away from Salt Lake City, UT and subsequently returned. Here, we describe three case studies examining: individual travelers (n = 4), paired travelers (n = 2), and a traveler (n = 1) who repeatedly left and returned. Our case studies examined the influences of dietary and drinking water inputs on fingernail stable isotope profiles for reconstructing travel histories. Stable isotope data were compared with theoretical and predictive models that laid the groundwork for anthropological and forensic geospatial reconstruction of travel histories. Drinking water inputs strongly influenced stable isotope profiles and the patterns observed were consistent with reported travel histories. Finally, observed fingernail stable isotope ratio data were consistent with modeled rates of stable isotope turnover and transition to expected values for the new environment. The results of our study further support the use of fingernail tissues for travel history reconstruction.     

USGS42 and USGS43: human-hair stable hydrogen and oxygen isotopic reference materials and analytical methods for forensic science and implications for published measurement results

Because there are no internationally distributed stable hydrogen and oxygen isotopic reference materials of human hair, the U.S. Geological Survey (USGS) has prepared two such materials, USGS42 and USGS43. These reference materials span values commonly encountered in human hair stable isotope analysis and are isotopically homogeneous at sample sizes larger than 0.2 mg. USGS42 and USGS43 human-hair isotopic reference materials are intended for calibration of δ(2)H and δ(18)O measurements of unknown human hair by quantifying (1) drift with time, (2) mass-dependent isotopic fractionation, and (3) isotope-ratio-scale contraction. While they are intended for measurements of the stable isotopes of hydrogen and oxygen, they also are suitable for measurements of the stable isotopes of carbon, nitrogen, and sulfur in human and mammalian hair. Preliminary isotopic compositions of the non-exchangeable fractions of these materials are USGS42(Tibetan hair)δ(2)H(VSMOW-SLAP) = -78.5 ± 2.3‰ (n = 62) and δ(18)O(VSMOW-SLAP) = +8.56 ± 0.10‰ (n = 18) USGS42(Indian hair)δ(2)H(VSMOW-SLAP) = -50.3 ± 2.8‰ (n = 64) and δ(18)O(VSMOW-SLAP) = +14.11 ± 0.10‰ (n = 18). Using recommended analytical protocols presented herein for δ(2)H(VSMOW-SLAP) and δ(18)O(VSMOW-SLAP) measurements, the least squares fit regression of 11 human hair reference materials is δ(2)H(VSMOW-SLAP) = 6.085δ(2)O(VSMOW-SLAP) - 136.0‰ with an R-square value of 0.95. The δ(2)H difference between the calibrated results of human hair in this investigation and a commonly accepted human-hair relationship is a remarkable 34‰. It is critical that readers pay attention to the δ(2)H(VSMOW-SLAP) and δ(18)O(VSMOW-SLAP) of isotopic reference materials in publications, and they need to adjust the δ(2)H(VSMOW-SLAP) and δ(18)O(VSMOW-SLAP) measurement results of human hair in previous publications, as needed, to ensure all results on are on the same scales.     

Variation of δ2H, δ18O & δ13C in crude palm oil from different regions in Malaysia: Potential of stable isotope signatures as a key traceability parameter

A total of 33 crude palm oil samples were randomly collected from different regions in Malaysia. Stable carbon isotopic composition (δ¹³C) was determined using Flash 2000 elemental analyzer while hydrogen and oxygen isotopic compositions (δ²H and δ¹⁸O) were analyzed by Thermo Finnigan TC/EA, wherein both instruments were coupled to an isotope ratio mass spectrometer. The bulk δ²H, δ¹⁸O and δ¹³C of the samples were analyzed by Hierarchical Cluster Analysis (HCA), Principal Component Analysis (PCA) and Orthogonal Partial Least Square-Discriminant Analysis (OPLS-DA). Unsupervised HCA and PCA methods have demonstrated that crude palm oil samples were grouped into clusters according to respective state. A predictive model was constructed by supervised OPLS-DA with good predictive power of 52.60%. Robustness of the predictive model was validated with overall accuracy of 71.43%. Blind test samples were correctly assigned to their respective cluster except for samples from southern region. δ¹⁸O was proposed as the promising discriminatory marker for discerning crude palm oil samples obtained from different regions. Stable isotopes profile was proven to be useful for origin traceability of crude palm oil samples at a narrower geographical area, i.e. based on regions in Malaysia. Predictive power and accuracy of the predictive model was expected to improve with the increase in sample size. Conclusively, the results in this study has fulfilled the main objective of this work where the simple approach of combining stable isotope analysis with chemometrics can be used to discriminate crude palm oil samples obtained from different regions in Malaysia. Overall, this study shows the feasibility of this approach to be used as a traceability assessment of crude palm oils.     

Comparison of bulk and compound-specific δ13C isotope ratio analyses for the discrimination between cannabis samples

Five marijuana samples were compared using bulk isotope analysis compound-specific isotope ratio analysis of the extracted cannabinoids. Owing to the age of our cannabis samples, four of the five samples were compared using the isotope ratios of cannabinol (CBN), a stable degradation product of Δ(9)-tetrahydrocannabinol (THC). Bulk δ(13)C isotope analysis discriminated between all five samples at the 95% confidence level. Compound-specific δ(13)C isotope analysis could not distinguish between one pair of the five samples at the 95% confidence level. All the measured cannabinoids showed significant depletion in (13)C relative to bulk isotope values; the isotope ratios for THC, CBN, and cannabidiol were on average 1.6‰, 1.7‰, and 2.2‰ more negative than the bulk values, respectively. A more detailed investigation needs to be conducted to assess the degree fractionation between the different cannabinoids, especially after aging.     

Stable carbon and nitrogen isotope ratios of sodium and potassium cyanide as a forensic signature

Sodium and potassium cyanide are highly toxic, produced in large amounts by the chemical industry, and linked to numerous high-profile crimes. The U.S. Centers for Disease Control and Prevention has identified cyanide as one of the most probable agents to be used in a chemical terrorism event. We investigated whether stable C and N isotopic content of sodium and potassium cyanide could serve as a forensic signature for sample matching, using a collection of 65 cyanide samples. Upon analysis, a few of the cyanide samples displayed nonhomogeneous isotopic content associated with degradation to a carbonate salt and loss of hydrogen cyanide. Most samples had highly reproducible isotope content. Of the 65 cyanide samples, >95% could be properly matched based on C and N isotope ratios, with a false match rate <3%. These results suggest that stable C and N isotope ratios are a useful forensic signature for matching cyanide samples.     

Refining Stable Oxygen and Hydrogen Isoscapes for the Identification of Human Remains in Mississippi,

Isoscape refinement is an essential component for accurately predicting region‐of‐origin in forensic investigations involving isotope analysis of unidentified human remains. Stable oxygen (δ¹⁸O) and hydrogen (δ²H) isotopes were measured from 57 tap water samples collected across Mississippi to model refined isoscapes for the state. A tap water conversion equation, δ¹⁸O_tw=1.64 δ¹⁸Op?31.35, was developed for the southeastern USA to test the prediction accuracy of the δ¹⁸O_tw isoscape using individuals with known residential histories. A local Mississippi resident (USAFA‐134) was assigned with 90% probability to the correct region‐of‐origin reported by the participant. Assignments for Georgia residents (USAFA‐118 and USAFA‐205) had variable results, predicting USAFA‐118 from Mississippi and USAFA‐205 as a nonlocal resident. Stable isotope values often overlap geographically and a multi‐isotope approach should be used when narrowing region(s)‐of‐origin(s). This study demonstrates the utility of refining isoscapes and the importance of tissue calibration in prediction assignments of human remains.     

Sourcing Brazilian marijuana by applying IRMS analysis to seized samples

The stable carbon and nitrogen isotopic ratios were measured in marijuana samples (Cannabis sativa L.) seized by the law enforcement officers in the three Brazilian production sites: Pernambuco and Bahia (the country's Northeast known as Marijuana Polygon), Pará (North or Amazon region) and Mato Grosso do Sul (Midwest). These regions are regarded as different with respect to climate and water availability, factors which impact upon the isotope fractionations of these elements within plants. It was possible to differentiate samples from the dry regions (Marijuana Polygon) from those from Mato Grosso do Sul and Pará, that present heavier rainfall. The results were in agreement with the climatic conditions of the suspected regions of origin and this demonstrates that seized samples can be used to identify the isotopic signatures of marijuana from the main producing regions in Brazil.     

Paired stable carbon and oxygen isotope analyses of human enamel for forensic human geolocation: An exploratory study

Stable carbon isotope analysis has proven utility for reconstructing dietary information in humans in past populations. The usefulness of stable carbon for forensic geolocation has been little investigated, largely because of the globalization of the human diet seemingly rendering it inconsequential. This study queried this assumption at a country-wide level on a known sample group. Stable carbon isotope values were obtained from human enamel with known biographical and geographical information to determine whether stable carbon, when paired with stable oxygen isotope values, could differentiate Canadians from non-Canadians. Samples originating outside of Canada were separated into three regions, and a linear discriminant analysis was used to generate discriminant functions that best separate the regions according to the stable carbon and oxygen isotope values. The results revealed two functions, where the first function explained 92.1% and the second 7.9% of the variance. Although some overlap in stable carbon and oxygen values was observed for individuals from both the United States and Canada, differences were observed between those from Canada and other geographical regions. This study demonstrated that pairing the dietary isotope carbon with the geolocator stable oxygen isotope, produced an interesting separation geographically, one that might well be helpful when attempting a geolocation query on unknown human remains.     

Detection of counterfeit antiviral drug Heptodin™ and classification of counterfeits using isotope amount ratio measurements by multicollector inductively coupled plasma mass spectrometry (MC-ICPMS) and isotope ratio mass spectrometry (IRMS)

Isotope ratio mass spectrometry (IRMS) and multicollector inductively coupled plasma mass spectrometry (MC-ICP-MS) are highly important techniques that can provide forensic evidence that otherwise would not be available. MC-ICP-MS has proved to be a very powerful tool for measuring high precision and accuracy isotope amount ratios. In this work, the potential of combining isotope amount ratio measurements performed by MC-ICP-MS and IRMS for the detection of counterfeit pharmaceutical tablets has been investigated.An extensive study for the antiviral drug Heptodin™ has been performed for several isotopic ratios combining MC-ICP-MS and an elemental analyser EA-IRMS for stable isotope amount ratio measurements. The study has been carried out for 139 batches of the antiviral drug and analyses have been performed for C, S, N and Mg isotope ratios. Authenticity ranges have been obtained for each isotopic system and combined to generate a unique multi-isotopic pattern only present in the genuine tablets. Counterfeit tablets have then been identified as those tablets with an isotopic fingerprint outside the genuine isotopic range.The combination of those two techniques has therefore great potential for pharmaceutical counterfeit detection. A much greater power of discrimination is obtained when at least three isotopic systems are combined. The data from these studies could be presented as evidence in court and therefore methods need to be validated to support their credibility. It is also crucial to be able to produce uncertainty values associated to the isotope amount ratio measurements so that significant differences can be identified and the genuineness of a sample can be assessed.