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.     

A City‐wide Investigation of the Isotopic Distribution and Source of Tap Waters for Forensic Human Geolocation Ground‐truthing

Human geolocation is prefaced on the accuracy of the geographic precision of mapped isotopic values for drinking water. As most people live in cities, it becomes important to understand city water supplies and how the isotopic values uniquely reflect that city. This study investigated the isotopic distribution of δ²H and δ¹⁸O from sourced tap waters that were collected from across the Metro Vancouver (MV) area (n = 135). The results revealed that the isotopic values reflect their water sources with a range of 5.3‰ for δ¹⁸O_tap and 29.3‰ for δ²H_tap for MV. The results indicate that individual cities need higher resolution studies to determine their tap water isotopic ranges, and a good understanding of the water supply network itself for human geolocation work. With an extended high‐resolution understanding of each city, human tissue may be compared with more certainty for geolocation.     

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.     

The Potential of Isotope Ratio Mass Spectrometry (IRMS) and Gas Chromatography‐IRMS Analysis of Triacetone Triperoxide in Forensic Explosives Investigations

Studying links between triacetone triperoxide (TATP) samples from crime scenes and suspects can assist in criminal investigations. Isotope ratio mass spectrometry (IRMS) and gas chromatography (GC)‐IRMS were used to measure the isotopic compositions of TATP and its precursors acetone and hydrogen peroxide. In total, 31 TATP samples were synthesized with different raw material combinations and reaction conditions. For carbon, a good differentiation and a linear relationship were observed for acetone–TATP combinations. The extent of negative (δ¹³C) fractionation depended on the reaction yield. Limited enrichment was observed for the hydrogen isotope (δ²H) values of the TATP samples probably due to a constant exchange of hydrogen atoms in aqueous solution. For oxygen (δ¹⁸O), the small isotopic range and excess of water in hydrogen peroxide resulted in poor differentiation. GC‐IRMS and IRMS data were comparable except for one TATP sample prepared with high acid concentration demonstrating the potential of compound‐specific isotope analysis. Carbon IRMS has practical use in forensic TATP investigations.     

Temporal variation of oxygen isotope ratios (δ18O) in drinking water: implications for specifying location of origin with human scalp hair

Previous work suggests that δ(18)O values of human hair can be used to constrain the region-of-origin of unknown individuals, but robust assessments of uncertainties in this method are lacking. Here we assess one source of uncertainty - temporal variation in the δ(18)O value of drinking water - using a monthly tap water survey of δ(18)O to develop geospatial models (i.e., maps) of the intra-annual variation (seasonality) in tap water δ(18)O for the contiguous USA. Temporal variation in tap water δ(18)O was correlated with water-supply type, and was related to geographic patterns of precipitation δ(18)O seasonality and water residence time. The maps were applied in a Bayesian framework to identify the geographic origin of an unidentified woman found in Utah, based on measured δ(18)O of scalp hair. The results are robust in specifying parts of the western USA as the most likely region-of-origin. Incorporation of tap water δ(18)O seasonality in the analysis reduces the precision of geographic assignments, but other sources of uncertainty (e.g., spatial interpolation uncertainty) have an equal or larger effect.     

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.     

Discrimination of Geographical Origin of Asian Garlic Using Isotopic and Chemical Datasets under Stepwise Principal Component Analysis

Isotopic compositions of δ²H, δ¹⁸O, δ¹³C, and δ¹⁵N and concentrations of 22 trace elements from garlic samples were analyzed and processed with stepwise principal component analysis (PCA) to discriminate garlic's country of origin among Asian regions including South Korea, Vietnam, Taiwan, and China. Results indicate that there is no single trace‐element concentration or isotopic composition that can accomplish the study's purpose and the stepwise PCA approach proposed does allow for discrimination between countries on a regional basis. Sequentially, Step‐1 PCA distinguishes garlic's country of origin among Taiwanese, South Korean, and Vietnamese samples; Step‐2 PCA discriminates Chinese garlic from South Korean garlic; and Step‐3 and Step‐4 PCA, Chinese garlic from Vietnamese garlic. In model tests, countries of origin of all audit samples were correctly discriminated by stepwise PCA. Consequently, this study demonstrates that stepwise PCA as applied is a simple and effective approach to discriminating country of origin among Asian garlics.     

Can Stable Isotopes and Radiocarbon Dating Provide a Forensic Solution for Curbing Illegal Harvesting of Threatened Cycads?

Cycads in South Africa are facing an extinction crisis due to the illegal extraction of plants from the wild. Proving wild origin of suspect ex situ cycads to the satisfaction of a court of law is difficult, limiting law enforcement efforts. We investigated the feasibility of using multiple stable isotopes to identify specimens removed from the wild. Relocated and wild specimens from two species in the African genus Encephalartos (E. lebomboensis and E. arenarius) were sampled. ¹⁴C analysis indicated that a ± 30‐year chronology could be reliably obtained from the cycads. For E. arenarius, pre‐relocation tissue was consistent with a wild origin, whereas tissue grown post‐relocation was isotopically distinct from the wild for ⁸⁷Sr/⁸⁶Sr and δ¹⁵N. For E. lebomboensis, δ³⁴S, δ¹⁸O, and ⁸⁷Sr/⁸⁶Sr were different between relocated and control plants, consistent with the >30 years since relocation. Our findings demonstrate the potential for a forensic isotope approach to identify illegal ex situ cycads.     

Trichophyton Mentagrophytes Perforates Hair of Adult Corpses in the Gaseous Period*

Abstract: Despite the substantial literature on mycology, there are still limited reports of the interaction between fungi and human hosts in the postmortem period. Thus, the main goal of this study was to investigate the in vitro perforation test using Trichophyton mentagrophytes on hair from adult corpses in the postmortem period (gaseous period). The protocol was carried out with positive (prepubescent children’s hair) and negative controls (healthy adult hair) as well. One strain of Trichophyton rubrum was also used as a negative perforation control. Perforations were found in all the hair samples from corpses and prepubescent children after 12–14 days exposure to T. mentagrophytes and were absent in the hair samples of healthy adults. Furthermore, hair perforation was not observed with T. rubrum. Our preliminary findings suggest the use of T. mentagrophytes as a potential marker of the death interval in forensic science.     

Case of Fatal Starvation: Can Stable Isotope Analysis Serve to Support Morphological Diagnosis and Approximate the Length of Starvation?

The diagnosis of death as a result of starvation is established on anthropological measurements, visual appearance of the deceased on external and internal examination, microscopic analysis, laboratory testing, and exclusion of other causes of death. Herein, we present our findings on a case of 95‐year‐old man who died of starvation. After the diagnosis of starvation was established by traditional forensic medicine methods, we have conducted retrospective segmental analysis of stable carbon (δ¹³C) and nitrogen (δ¹⁵N) isotope ratios in hair sample. This method reveals periods of starvation through decrease in δ¹³C and increase in δ¹⁵N along the strand of hair. Our analysis revealed the decrease of 0.6 ‰ in δ¹³C during the last 10–12 weeks prior to death, similar as reported in other investigations. Also, a decrease of 0.7 ‰ in δ¹⁵N during the last 8–10 weeks prior to death was determined that was different than observed in previous studies.     

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.     

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.     

Results of four inter-laboratory comparisons provided by the Forensic Isotope Ratio Mass Spectrometry (FIRMS) network

Between 2004 and 2008 the Forensic Isotope Ratio Mass Spectrometry (FIRMS) network organised four Inter-Laboratory Camparison excercises with the aim of harmonising and validating isotopic measurements to be used for forensic applications.The samples distributed comprised materials of potential forensic interest such as packaging and pharmaceuticals to be analysed for δ²H, δ¹³C, δ¹⁵N and δ¹⁸O composition. As many as 30 international laboratories participated in these comparisons. Results have shown overall improvements for both within- and inter-laboratory reproducibility with respect to δ¹³C and δ¹⁵N measurements.Only a small number of laboratories returned results for δ²H and δ¹⁸O analysis and these results highlighted a need for improvement in the reproducibility of these measurements and a need to address the exchange of hydrogen between samples and ambient moisture. Results also highlighted the importance of sample preparation procedures and the need to standardise both these procedures and calibration against Standard Reference Materials. Future Inter-Laboratory Comparison exercises will assess the suitability of laboratories to submit data to national and international databases similar to those currently operated for fingerprints, DNA etc.     

Carbon and Nitrogen Stable Isotope Analyses of Ephedra Plant and Ephedrine Samples and Their Application For Methamphetamine Profiling

In this study, stable isotope ratio analysis was used to track the precursor information of methamphetamine. The δ¹³C and δ¹⁵N values of 30 nature ephedra plants, 12 synthetic ephedrine/pseudoephedrine (ephedrine), 14 natural ephedrine, and 987 seized methamphetamine samples were measured and compared. Due to different weather and earth conditions, the δ¹³C and δ¹⁵N values of ephedra plants grown in the east and the west of Inner Mongolia showed great difference. The δ¹⁵N values of ephedra plants were consistent with related ephedrine extracted from them. Moreover, the criteria to infer the synthetic origin of ephedrine were set up after the analysis of natural and synthetic ephedrine samples. Finally, the precursor origins of 949 seized methamphetamine samples synthesized by Emde and Nagai method were tentatively inferred. Influenced by different preprecursors, the δ¹³C values of seized methamphetamine samples that synthesized from P2P also showed great difference, and this result is consistent with the reported data.     

Stable Isotope Ratios of Marijuana. I. Carbon and Nitrogen Stable Isotopes Describe Growth Conditions*

Abstract: There remains significant uncertainty in illicit marijuana cultivation. We analyzed the δ¹³C and δ¹⁵N of 508 domestic samples from known U.S.A. counties, 31 seized from a single location, 5 samples grown in Mexico and Colombia, and 10 northwest border seizures. For a subset, inflorescences and leaves were analyzed separately. These data revealed a strong correspondence, with inflorescences having slightly higher δ¹³C and δ¹⁵N values than leaves. A framework for interpreting these results is introduced and evaluated. Samples identified as outdoor‐grown by δ¹³C were generally recorded as such by the Drug Enforcement Administration (DEA). DEA‐classified indoor‐grown samples had the most negative δ¹³C values, consistent with indoor cultivation, although many were also in the outdoor‐grown domain. δ¹⁵N indicated a wide range of fertilizers across the dataset. Samples seized at the single location suggested multiple sources. Northwest border δ¹³C values suggested indoor growth, whereas for the Mexican and Colombian samples they indicated outdoor growth.     

Does Mirror Imaging a Radiograph Affect Reliability of Age Assessment Using the Greulich and Pyle Atlas?*

Abstract: Age estimation is routinely undertaken by comparing radiographs of the individual in question to published reference samples of individuals of known age. This study examines the reliability of age estimation utilizing the Greulich and Pyle atlas in relation to both left‐ and right‐hand/wrist radiographs and explores whether reversing right‐hand/wrist radiographs, so that they are in the same anatomical orientation as those images used in the atlas affects reliability. A total of 403 left‐hand/wrist radiographs and 415 right‐hand/wrist radiographs were age assessed using the Greulich and Pyle atlas. Analysis showed that there is no significant loss in reliability when radiographs of the right hand (women R² = 0.887 and men R² = 0.907) are utilized instead of the left (women R² = 0.939 and men R² = 0.940) or when they are assessed as mirror images to those printed in the reference atlas (reversed female left hand R² = 0.929 and reversed male left hand R² = 0.931).     

Development of a Protein‐based Human Identification Capability from a Single Hair

Shed human hair (lacking root nuclear DNA) frequently contributes important information to forensic investigations involving human identification. Detection of genetic variation observed in amino acid sequences of hair proteins provides a new suite of identity markers that augment microscopic hair analysis and mitochondrial DNA sequencing. In this study, a new method that completely dissolves single hairs using a combination of heat, ultrasonication, and surfactants was developed. Dissolved proteins were digested and genetically variant peptide (GVP) profiles were obtained for single hairs (25 mm) via high‐resolution nanoflow liquid chromatography‐based mass spectrometry and a novel exome‐driven bioinformatic approach. Overall, 6519 unique peptides were identified and a total of 57 GVPs were confirmed. Random match probabilities ranged between 2.6 × 10^?2 and 6.0 × 10^?9. The new bioinformatic strategy and ability to analyze GVPs in forensically relevant samples sizes demonstrate applicability of this approach to distinguish individuals in forensic contexts.     

Age Estimation Based on DNA Methylation Using Blood Samples From Deceased Individuals

Age estimation using DNA methylation levels has been widely investigated in recent years because of its potential application in forensic genetics. The main aim of this study was to develop an age predictor model (APM) for blood samples of deceased individuals based in five age-correlated genes. Fifty-one samples were analyzed through the bisulfite polymerase chain reaction (PCR) sequencing method for DNA methylation evaluation in genes ELOVL2, FHL2, EDARADD, PDE4C, and C1orf132. Linear regression was used to analyze relationships between methylation levels and age. The model using the highest age-correlated CpG from each locus revealed a correlation coefficient of 0.888, explaining 76.3% of age variation, with a mean absolute deviation from the chronological age (MAD) of 6.08 years. The model was validated in an independent test set of 19 samples producing a MAD of 8.84 years. The developed APM seems to be informative and could have potential application in forensic analysis.     

Differentiation of Morphologically Similar Human Head Hairs from Two Demographically Similar Individuals Using Amino Acid Ratios,

Human hair is frequently encountered as forensic evidence and can contribute valuable information to investigators. Conventional forensic hair analyses include microscopic hair comparison (MHC) and DNA analysis. However, MHC is not supported by statistics and DNA analysis cannot always be performed. Recent studies have demonstrated that evaluation of differences in the hair proteins may offer an alternate method to these analyses. In this study, an evaluation of the amino acids present in hair was investigated as an approach to differentiate morphologically indistinguishable hair samples from two demographically similar individuals. Proteins in the hair were digested using hydrochloric acid, and the resulting amino acids were derivatized with N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) for analysis using gas chromatography-mass spectrometry (GC-MS). Eight derivatized amino acids were detected and quantified relative to an internal standard, L-norvaline, and used to construct twenty-eight amino acid ratios. Hair samples were collected from four areas of the head on various days over the course of one month, and no significant differences in amino acid ratios (p-value > 0.05) were observed among the areas of the head, and the ratios were consistent over the time period of this study. Additionally, fifteen of these amino acid ratios were found to be significantly different between the two individuals when compared using a two-sample t-test (p-value ≤ 0.05). These data indicate that amino acid analysis was able to differentiate two morphologically similar hair samples from different individuals and demonstrates the applicability of this method to distinguish similar hair samples when DNA analysis cannot be performed.     

Forensic applications of stable isotope analysis: Case studies of the origins of water in mislabeled beer and contaminated diesel fuel

This paper describes the use of oxygen (¹⁸O) isotope analysis of water contained in two different materials — beer and diesel fuel — involved in the resolution of two separate cases. In the first case study, it was possible to demonstrate that a sample of beer labelled as premium brand in fact belonged to a cheap brand. The second case related to the contamination of diesel fuel from a service station. The diesel fuel contained visible amounts of water, which caused vehicles that had been filled up with it to become defective. For insurance purposes, it was necessary to determine the source of water. The δ¹⁸O values for the water of nearly all samples of diesel was close to the δ¹⁸O of local tap water at the filling station.