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.     

Stable isotope analysis of white paints and likelihood ratios

Architectural paints are commonly found as trace evidence at scenes of crime. Currently the most widely used technique for the analysis of architectural paints is Fourier Transformed Infra-Red Spectroscopy (FTIR). There are, however, limitations to the forensic analysis of white paints, and the ability to discriminate between samples.Isotope ratio mass spectrometry (IRMS) has been investigated as a potential tool for the analysis of architectural white paints, where no preparation of samples prior to analysis is required. When stable isotope profiles (SIPs) are compared, there appears to be no relationship between paints from the same manufacturer, or between paints of the same type. Unlike existing techniques, IRMS does not differentiate resin samples solely on the basis of modifier or oil-type, but exploits additional factors linked to samples such as geo-location where oils added to alkyd formulations were grown. In combination with the use of likelihood ratios, IRMS shows potential, with a false positive rate of 2.6% from a total of 1275 comparisons.     

On the use of IRMS in forensic science: proposals for a methodological approach

The flourishing number of publications on the use of isotope ratio mass spectrometry (IRMS) in forensic science denotes the enthusiasm and the attraction generated by this technology. IRMS has demonstrated its potential to distinguish chemically identical compounds coming from different sources. Despite the numerous applications of IRMS to a wide range of forensic materials, its implementation in a forensic framework is less straightforward than it appears. In addition, each laboratory has developed its own strategy of analysis on calibration, sequence design, standards utilisation and data treatment without a clear consensus. Through the experience acquired from research undertaken in different forensic fields, we propose a methodological framework of the whole process using IRMS methods. We emphasize the importance of considering isotopic results as part of a whole approach, when applying this technology to a particular forensic issue. The process is divided into six different steps, which should be considered for a thoughtful and relevant application. The dissection of this process into fundamental steps, further detailed, enables a better understanding of the essential, though not exhaustive, factors that have to be considered in order to obtain results of quality and sufficiently robust to proceed to retrospective analyses or interlaboratory comparisons.     

Microbial metagenome profiling using amplicon length heterogeneity-polymerase chain reaction proves more effective than elemental analysis in discriminating soil specimens

The combination of soil's ubiquity and its intrinsic abiotic and biotic information can contribute greatly to the forensic field. Although there are physical and chemical characterization methods of soil comparison for forensic purposes, these require a level of expertise not always encountered in crime laboratories. We hypothesized that soil microbial community profiling could be used to discriminate between soil types by providing biological fingerprints that confer uniqueness. Three of the six Miami-Dade soil types were randomly selected and sampled. We compared the microbial metagenome profiles generated using amplicon length heterogeneity-polymerase chain reaction analysis of the 16S rRNA genes with inductively coupled plasma optical emission spectroscopy analysis of 13 elements (Al, B, Ca, Cu, Fe, K, Mg, Mn, Na, P, S, Si, and Zn) that are commonly encountered in soils. Bray-Curtis similarity index and analysis of similarity were performed on all data to establish differences within sites, among sites, and across two seasons. These data matrices were used to group samples that shared similar community patterns using nonmetric multidimensional scaling analysis. We concluded that while chemical characterization could provide some differentiation between soils, microbial metagenome profiling was better able to discriminate between the soil types and had a high degree of reproducibility, therefore proving to be a potential tool for forensic soil comparisons.     

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.     

Forensic analysis of explosives using isotope ratio mass spectrometry (IRMS) — Preliminary study on TATP and PETN

The application of isotopic techniques to investigations requiring the provision of evidence to a Court is limited. The objective of this research was to investigate the application of light stable isotopes and isotope ratio mass spectrometry (IRMS) to solve complex forensic cases by providing a level of discrimination not achievable utilising traditional forensic techniques.Due to the current threat of organic peroxide explosives, such as triacetone triperoxide (TATP), research was undertaken to determine the potential of IRMS to differentiate samples of TATP that had been manufactured utilising different starting materials and/or manufacturing processes. In addition, due to the prevalence of pentaerythritoltetranitrate (PETN) in detonators, detonating cord, and boosters, the potential of the IRMS technique to differentiate PETN samples from different sources was also investigated.Carbon isotope values were measured in fourteen TATP samples, with three definite groups appearing in the initial sample set based on the carbon data alone. Four additional TATP samples (in a second set of samples) were distinguishable utilising the carbon and hydrogen isotopic compositions individually, and also in combination with the oxygen isotope values. The 3D plot of the carbon, oxygen and hydrogen data demonstrated the clear discrimination of the four samples of TATP. The carbon and nitrogen isotope values measured from fifteen PETN samples, allowed samples from different sources to be readily discriminated.This paper demonstrates the successful application of IRMS to the analysis of explosives of forensic interest to assist in discriminating samples from different sources. This research represents a preliminary evaluation of the IRMS technique for the measurement of stable isotope values in TATP and PETN samples, and supports the dedication of resources for a full evaluation of this application in order to achieve Court reportable IRMS results.     

Simultaneous identification and δ¹³C classification of drugs using GC with concurrent single quadrupole and isotope ratio mass spectrometers

In this study, δ13C values of six cocaine samples were identified and classified using a single quadrupole mass spectrometer and an isotope ratio mass spectrometry (IRMS) as simultaneous gas chromatography detectors. Our instrument modification is simple to use and is useful (i) when the sample is of limited size or can only be injected once, (ii) to help identify peaks in a complicated IRMS chromatogram, and (iii) to help differentiate very simple systems when impurity profiling is not possible. The EI-MS confirmed the identity of cocaine in each sample. The IRMS data distinguished 12 of the 15 possible pair-wise comparisons at the 95% CL. Three samples could not be differentiated by their δ13C ratios for cocaine. ANOVA demonstrated that the measurement variance was consistently larger than the sample variance. As the δ13C values clearly show, this technique enables the exclusion of a potential common source even when two samples have otherwise identical chemical and physical properties.     

The role of isotope ratio mass spectrometry as a tool for the comparison of physical evidence

This paper considers how likelihood ratios can be derived for a combination of physical, chemical and isotopic measurements. Likelihood ratios were formulated based on the characteristics of a small convenience sample of 20 duct tapes. The propositions considered were:

• H_ptwo samples are from the same batch
• H_dtwo samples are from different batches.

The physical and isotopic characteristics of ten rolls of duct tape were shown to be consistent throughout each roll.The width and thickness of the tapes and the density of the scrim fibres provided equivalent information and the combined physical characteristics provided a basis upon which to discriminate between many of the samples.Scatter-plots and confidence ellipses provided a convenient method to group the isotopic composition of the tape backing material and provided a basis to discriminate between samples which were physically indistinguishable. Considering both the physical and isotopic characteristics it was possible, at best, to ascertain that the evidence provided moderately strong support for the proposition that two samples of tape were derived from the same batch (LR = 400).Kernel density estimates were used to model the distribution of isotopic compositions of the backing material. Using this technique it was possible to estimate objectively the probability that a sample with given characteristics could be drawn, at random, from the background population and to calculate a likelihood ratio based on the propositions above.The strength of evidence which could be presented by either model was ultimately limited by the size of the background sample.     

Stable isotope profiling of burnt wooden safety matches

Arson is a significant problem around the world, and is a crime which results in a low number of convictions. The scene of an arson can be varied, commercial, residential or national park, and recently cases have been identified which were initiated by a lit match. Matches can be recovered from a scene, usually in a burnt condition. The benefit of analysing unburnt matches has been researched previously [1,2]. In most cases, burnt matches are recovered from scenes, and therefore the research was extended to investigate the potential of using IRMS to analyse burnt matches. This includes samples which have been exposed to petrol, and various fire extinguishing chemicals.Matches were sectioned to reveal central unburnt portions of wood and analysed by isotope ratio mass spectrometry (IRMS). The stable isotope profile (SIP) of the wooden matchstick samples was unaffected by the presence of both petrol and a variety of fire extinguisher chemicals. Any changes seen could be attributed to the natural variability of isotopic composition encountered in a natural material such as wood. These findings were confirmed by the isotope analysis of 19 matchstick samples placed in mock fire training scenarios. The data was examined using a paired t-test and Hotellings T² test for a single sample.     

Background survey of polyethylene in the Australian Capital Territory – A demonstration of variability in isotopic abundance values and their application to forensic casework

Plastics including adhesive tapes, cable ties, and packaging are common evidence types encountered in forensic investigations and casework. Traditional examination techniques such as Fourier Transform Infrared (FTIR) spectroscopy lack specificity and are unable to discern differences within the same polymer structures leaving the analyst with a generic identification. High quality manufacturing methods further amplify the limitations in detecting variability between samples. Isotope Ratio Mass Spectrometry (IRMS) has been shown to be a valuable technique in further discriminating plastics. Discrimination is achieved by analysing the relative abundances of stable isotopes within a sample, with differences detected in isotope ratios possibly attributed to the source of raw materials and fractionation during the manufacturing process. A survey of cling wraps and re-sealable zipper storage bags collected in the Australian Capital Territory was undertaken to assess the variability in carbon and hydrogen isotope ratios of different brands and samples. The results of this research are discussed, particularly with respect to within and between brand trends, and a case study is presented as an example of the value of including IRMS in a casework context.     

Discrimination of transparent polyethylene films based on identification of inorganic antiblocking agents

Several nondestructive and semidestructive analyses were used to discriminate colorless transparent polyethylene bags. Transparent plastic bags made from low-density and linear low-density polyethylene usually contain antiblocking agents to prevent sticking of the film, which makes it difficult to open the mouths of plastic bags. Inorganic antiblocking agents are uniformly dispersed in polyethylene films, so they are easily observed using optical microscopy. The particle size distributions of the antiblocking agents were compared by statistical tests. Particle composition was examined by elemental analysis using energy-dispersive X-ray spectrometry with a scanning electron microscope and by molecular vibration analysis using a sensitive microscopic Raman spectrometer. The compositions could be determined nondestructively on the basis of the results. These data reinforce the morphological discrimination. Morphological discrimination of the dispersed antiblocking agent powders, statistical comparison of particle size distributions, and compositional analysis of the antiblocking agents strengthen the ability to discriminate polyethylene films.     

The Measurement of Stable Carbon Isotope Ratios of Eight Methamidophos Samples

Between December 2007 and January 2008, people suffered from food poisoning in the Japanese prefectures of Chiba and Hyogo after eating frozen dumplings (gyoza) produced in China, which had very high concentrations (1490–19,290 ppm) of methamidophos (O,S‐dimethyl phosphoramidothioate). Thus, we measured the stable carbon isotope ratio of methamidophos using GC/C/IRMS to identify the source. We analyzed seven methamidophos reagents and one Chinese agricultural methamidophos chemical (MTD600) that contained many impurities. The δ¹³C values of the seven methamidophos reagents and MTD600 ranged from ?49.23‰ to ?31.90‰, with an average SD of 0.20‰, very high precision. This difference (17.33‰) was very large compared with that in previous reports and may be attributable to the material itself and the chemical processing of methamidophos. Criminals can easily obtain pesticides such as methamidophos; therefore, it is very important to identify the pesticide source and distribution route using stable isotopic science in the future.     

Forensic analysis of explosives using isotope ratio mass spectrometry (IRMS) — Discrimination of ammonium nitrate sources

An evaluation was undertaken to determine if isotope ratio mass spectrometry (IRMS) could assist in the investigation of complex forensic cases by providing a level of discrimination not achievable utilising traditional forensic techniques. The focus of the research was on ammonium nitrate (AN), a common oxidiser used in improvised explosive mixtures.The potential value of IRMS to attribute Australian AN samples to the manufacturing source was demonstrated through the development of a preliminary AN classification scheme based on nitrogen isotopes. Although the discrimination utilising nitrogen isotopes alone was limited and only relevant to samples from the three Australian manufacturers during the evaluated time period, the classification scheme has potential as an investigative aid.Combining oxygen and hydrogen stable isotope values permitted the differentiation of AN prills from three different Australian manufacturers. Samples from five different overseas sources could be differentiated utilising a combination of the nitrogen, oxygen and hydrogen isotope values. Limited differentiation between Australian and overseas prills was achieved for the samples analysed.The comparison of nitrogen isotope values from intact AN prill samples with those from post-blast AN prill residues highlighted that the nitrogen isotopic composition of the prills was not maintained post-blast; hence, limiting the technique to analysis of un-reacted explosive material.     

Forensic Analysis of Explosives Using Isotope Ratio Mass Spectrometry (IRMS)—Part 2: Forensic Inter-Laboratory Trial: Bulk Carbon and Nitrogen Stable Isotopes in a Range of Chemical Compounds (Australia and New Zealand)

Abstract:  Comparability of data over time and between laboratories is a key issue for consideration in the development of global databases, and more broadly for quality assurance in general. One mechanism that can be utilized for evaluating traceability is an inter-laboratory trial. This paper addresses an inter-laboratory trial conducted across a number of Australian and New Zealand isotope ratio mass spectrometry (IRMS) laboratories. The main objective of this trial was to determine whether IRMS laboratories in these countries would record comparable values for the distributed samples. Four carbon containing and four nitrogen containing compounds were distributed to seven laboratories in Australia and one in New Zealand. The laboratories were requested to analyze the samples using their standard procedures. The data from each laboratory was evaluated collectively using International Standard ISO 13528 ( Statistical methods for use in proficiency testing by inter-laboratory comparisons ). "Warning signals" were raised against one participant in this trial. "Action signals" requiring corrective action were raised against four participants. These participants reviewed the data and possible sources for the discrepancies. This inter-laboratory trial was successful in providing an initial snapshot of the potential for traceability between the participating laboratories. The statistical methods described in this article could be used as a model for others needing to evaluate stable isotope results derived from multiple laboratories, e.g., inter-laboratory trials/proficiency testing. Ongoing trials will be conducted to improve traceability across the Australian and New Zealand IRMS community.     

Identification of a disinterred grave by molecular and stable isotope analysis

Confirmation of a potential disinterred grave was sought by GC and GC/MS analyses of lipid extracts of whole soils and white particulate matter. Fatty acid profiles and concentrations determined for three of the soils correlated with the deposition of a large amount of exogenous organic matter, most likely adipocere and/or decomposed body fat. Determination of C_16:0 and C_18:0 fatty acid δ¹³C values by GC/C/IRMS revealed the input to be isotopically distinct from common British domesticated animals, plotting closely to values determined for adipose fat obtained from of a murder victim. By considering the difference between δ¹³C values (Δ¹³C_18:0–16:0) a potential isotopic proxy for identifying the source of adipocere (human) and adipose tissue was proposed.     

Qualitative Differences Among Rural and Urban Intimate Violence Victimization Experiences and Consequences: A Pilot Study

Relatively little is known about rural women's intimate violence experiences in comparison to urban women's experiences, partly because of the difficulty in accessing rural women. This pilot study used a protective order sample of 23 women (15 urban and 8 rural), which provides an access point that is relatively similar for comparisons across rural and urban areas. The number of participants is low and, therefore, results are preliminary. However, several significant findings emerged. Rural women reported significantly less social support, less education, less income, more physical abuse in the preceding year, more childhood physical and sexual abuse, and worse overall health and mental health, as well as encountering abuse earlier in the relationship. Both groups reported higher rates of illegal drug and cigarette use than those among the general population. The findings highlight some overall important themes in examining rural and urban intimate violence victims by suggesting that rural and urban intimate violence victims have different victimization experiences and service needs. Implications for further research and intervention are discussed.     

Comparison of the Columnar‐Thin‐Film and Vacuum‐Metal‐Deposition Techniques to Develop Sebaceous Fingermarks on Nonporous Substrates,

Both the columnar‐thin‐film (CTF) and the vacuum‐metal‐deposition (VMD) techniques for visualizing sebaceous fingermarks require the deposition of a material thereon in a vacuum chamber. Despite that similarity, there are many differences between the two techniques. The film deposited with the CTF technique has a columnar morphology, but the film deposited with the VMD technique comprises discrete islands. A split‐print methodology on a variety of fingermarked substrates was used to determine that the CTF technique is superior for developing fingermarks on clear sandwich bags and partial bloody fingermarks on stainless steel. Both techniques are similar in their ability to develop fingermarks on glass but the CTF technique yields higher contrast. The VMD technique is superior for developing fingermarks on white grocery bags and the smooth side of Gloss Finish Scotch Multitask^? tape. Neither technique worked well for fingermarks on black garbage bags.     

A case study on the application of isotope ratio mass spectrometry (IRMS) in determining the provenance of a rock used in an alleged nickel switching incident

The application of isotope ratio mass spectrometry (IRMS) in forensic science to establish the provenance of a range of questioned substances including soils, drugs, explosives, currency, ivory and rhino horn has been widely documented. The present study wishes to highlight the applicability of IRMS and specifically stable carbon IRMS in determining the provenance of a carbonate rock that was switched for nickel metal exported from South Africa to Israel. The technique employed effectively argued against a South African origin for the rock whilst simultaneously supporting an Israeli origin, enabling investigators to focus their attention accordingly. The study represents the first documented instance known to the authors where IRMS has been employed in the forensic geo-location of a rock.     

Gait analysis in forensic medicine*

Recordings from video surveillance systems are used as evidence from crime scenes. It would be useful to perform comparisons between disguised perpetrators and suspects based on their gait. We applied functional anatomical and biomechanical knowledge to analyze the gait of perpetrators, as recorded on surveillance video. Using a structured checklist, which addresses the single body segments during gait, we were able to give a statement concerning the gait patterns. Characteristic parameters were, e.g., varus instability in the knee at heel strike, and larger lateral flexion of the spinal column to one side than the other. Based on these characteristic features, we are able to state with reasonable certainty whether the suspect could be the perpetrator, but it is not possible to identify the perpetrator positively. Nevertheless, we have been involved in several cases where the court has found that this type of gait analysis was a valuable tool.