A concept study on identification and attribution profiling of chemical threat agents using liquid chromatography-mass spectrometry applied to Amanita toxins in food

Accidental or deliberate poisoning of food is of great national and international concern. Detecting and identifying potentially toxic agents in food is challenging due to their large chemical diversity and the complexity range of food matrices. A methodology is presented whereby toxic agents are identified and further characterized using a two-step approach. First, generic screening is performed by LC/MS/MS to detect toxins based on a list of selected potential chemical threat agents (CTAs). After identifying the CTAs, a second LC/MS analysis is performed applying accurate mass determination and the generation of an attribution profile. To demonstrate the potential of the methodology, toxins from the mushrooms Amanita phalloides and Amanita virosa were analyzed. These mushrooms are known to produce cyclic peptide toxins, which can be grouped into amatoxins, phallotoxins and virotoxins, where α-amanitin and β-amanitin are regarded as the most potent. To represent a typical complex food sample, mushroom stews containing either A. phalloides or A. virosa were prepared. By combining the screening method with accurate mass analysis, the attribution profile for the identified toxins and related components in each stew was established and used to identify the mushroom species in question. In addition, the analytical data was consistent with the fact that the A. virosa specimens used in this study were of European origin. This adds an important piece of information that enables geographic attribution and strengthens the attribution profile.     

Preliminary study of the succession pattern of necrobiont insects, colonising species and larvae on pig carcasses in Temuco (Chile) for forensic applications

Knowledge regarding the succession patterns of insects that visit carcasses as well as the other arthropod that colonise them and analysis of the parameters that are associated with larvae allow calculation of the minimum postmortem interval (PMI). This information is obtained from experiments carried out under specific geoclimatic conditions, which determine their application in forensic environments under similar conditions. The field study presented here is the first in Chile to analyse the decomposition process of pig carcasses and the associated succession of insects, colonising species and parameters related to larval masses. All of the larvae obtained from daily samples were measured (in mm), and their mean, range, standard deviation and stage of development (instars) were determined. The carcasses reached the dry remains stage in only 11 days. Seven species of Diptera visited the carcass during the process, but only two species colonised it by means of egg deposition followed by development of larvae: Cochliomyia macellaria (Fabricius) and Lucilia sericata (Meigen) (Diptera: Calliphoridae), which exhibited a duration of the development cycle from egg to adult of 21 days. The collected Coleoptera correspond to five predator species (Staphilinidae and Histeridae). From the results of this study, it can be concluded that only some of the insect species present in this region can provide information that can be used in forensic entomology and that analysis of larval masses of colonising species can be a valuable tool for determining the PMI(min) in this region of Chile.     

In vitro production of GHB in blood and serum samples under various storage conditions

The in vitro production of GHB was observed in freshly collected, untreated whole blood samples using glass BD-Vacutainers and polypropylene S-monovettes. GHB concentrations were determined daily over a period of one week and after 3, 6 and 9 weeks again. Furthermore, the GHB concentration in 40 untreated random whole blood samples stored at 4°C for a longer period of time (10 samples 12 month, 10 samples 24 month and 20 samples 36 month) was also determined. For comparison, the in vitro production of GHB in freshly collected and prepared serum samples was observed. GHB serum concentrations were determined three times over a period of one week and once again after six weeks. Sample preparation was performed by means of methanolic extraction following the precipitation of whole blood and serum samples. A methanolic standard calibration was done in a low range of 0.005-0.1 μg/mL (LOD: 0.004, LLOQ: 0.013). For quantification a spiked blood bank serum with a determined GHB concentration of 0.09 μg/mL was used. Corrected calibrations in the range of 0.09-5.09 μg/mL were used (LOD: 0.08 μg/mL, LLOQ: 0.30 μg/mL), recovery: 91.3% (high level: 4.09 μg/mL) 50.5% (low level: 0.19 μg/mL). RESULTS: Relevant elevation of GHB was observed in all whole blood samples stored in liquid form (4°C or room temperature). In two of the 40 whole blood samples stored over a longer period of time at 4°C, GHB concentrations in the range of 13 μg/mL were even determined. These findings constitute grounds for caution. Even a GHB cut-off level of 5 μg/mL cannot be considered as "absolutely positive" proof of a case of exogenous administration, at least in untreated liquid blood samples in long time storage. However, no significant elevations of GHB were otherwise observed in any of the serum samples independently of storage temperature nor in the whole blood samples that were frozen for storage. CONCLUSIONS: The results suggest that the cut-off for exogenous GHB of 5 μg/mL could be lowered significantly, with the consequence of winning valuable time for the potential victim, but only if serum is collected for GHB determination or if the whole blood sample is frozen immediately after collection and the procedure well documented.     

Raman spectroscopy and microspectrophotometry of reactive dyes on cotton fibres: Analysis and detection limits

A collaborative study on Raman spectroscopy and microspectrophotometry (MSP) was carried out by members of the ENFSI (European Network of Forensic Science Institutes) European Fibres Group (EFG) on different dyed cotton fabrics. The detection limits of the two methods were tested on two cotton sets with a dye concentration ranging from 0.5 to 0.005% (w/w). This survey shows that it is possible to detect the presence of dye in fibres with concentrations below that detectable by the traditional methods of light microscopy and microspectrophotometry (MSP). The MSP detection limit for the dyes used in this study was found to be a concentration of 0.5% (w/w). At this concentration, the fibres appear colourless with light microscopy. Raman spectroscopy clearly shows a higher potential to detect concentrations of dyes as low as 0.05% for the yellow dye RY145 and 0.005% for the blue dye RB221. This detection limit was found to depend both on the chemical composition of the dye itself and on the analytical conditions, particularly the laser wavelength. Furthermore, analysis of binary mixtures of dyes showed that while the minor dye was detected at 1.5% (w/w) (30% of the total dye concentration) using microspectrophotometry, it was detected at a level as low as 0.05% (w/w) (10% of the total dye concentration) using Raman spectroscopy. This work also highlights the importance of a flexible Raman instrument equipped with several lasers at different wavelengths for the analysis of dyed fibres. The operator and the set up of the analytical conditions are also of prime importance in order to obtain high quality spectra. Changing the laser wavelength is important to detect different dyes in a mixture.     

Serious head and neck injury as a predictor of occupant position in fatal rollover crashes

Serious head and neck injuries are a common finding in fatalities associated with rollover crashes. In some fatal rollover crashes, particularly when ejection occurs, the determination of which occupant was driving at the time of the crash may be uncertain. In the present investigation, we describe the analysis of rollover crash data from the National Automotive Sampling System-Crashworthiness Data System for the years 1997 through 2007 in which we examined the relationship between a serious head and neck injury in an occupant and a specified degree of roof deformation at the occupant's seating position. We found 960 occupants who qualified for the analysis, with 142 deaths among the subjects. Using a ranked composite head and neck injury score (the HNISS) we found a strong relationship between HNISS and the degree of roof crush. As a result of the analysis, we arrived at a predictive model, in which each additional unit increase in HNISS equated to an increased odds of roof crush as follows: for ≥8cm of roof crush compared with <8cm by 4%, for ≥15cm of roof crush compared to <8cm by 6% and for ≥30cm of roof crush compared to <8cm by 11%. We describe two hypothetical scenarios in which the model could be applied to the real world investigation of occupant position in a rollover crash-related fatality.     

Lucilia silvarum Meigen, 1826 (Diptera: Calliphoridae)-A new species of interest for forensic entomology in Europe

In Europe, the blowfly genus Lucilia is represented in Forensic Entomology mainly by the species L. ampullacea, L. caesar, L. illustris and L. sericata. In the US, Lucilia silvarum is rarely recorded as a carrion breeding species but usually as a more or less exclusive parasite of frogs and toads. We present three forensic cases from different European countries reporting, for the first time, L. silvarum on human bodies that were found close to lakes, wetlands, or riversides. To use this species for post-mortem interval estimations, thermal development data is needed. The first step is accurate identification by morphological and molecular means. Therefore, we analysed a 611bp part of the mitochondrial COI region for 23 specimens of L. silvarum from 9 different geographical regions, all of which give the same haplotype. Differences within the haplotype varied by up to 0.2%. Comparison between the haplotype found and those published on GenBank showed up to 1.2% variance. Moreover, we present an updated key for the morphological identification of the third larval instars of European Lucilia spp. of forensic importance, adding not only L. silvarum, but also L. cuprina which was recorded in Europe for the first time about 20 years ago.     

Fatal tolperisone poisoning: autopsy and toxicology findings in three suicide cases

Tolperisone (Mydocalm) is a centrally acting muscle relaxant with few sedative side effects that is used for the treatment of chronic pain conditions. We describe three cases of suicidal tolperisone poisoning in three healthy young subjects in the years 2006, 2008 and 2009. In all cases, macroscopic and microscopic autopsy findings did not reveal the cause of death. Systematic toxicological analysis (STA) including immunological tests, screening for volatile substances and blood, urine and gastric content screening by GC-MS and HPLC-DAD demonstrated the presence of tolperisone in all cases. In addition to tolperisone, only the analgesics paracetamol (acetaminophen), ibuprofen and naproxen could be detected. The blood ethanol concentrations were all lower than 0.10 g/kg. Tolperisone was extracted by liquid-liquid extraction using n-chlorobutane as the extraction solvent. The quantification was performed by GC-NPD analysis of blood, urine and gastric content. Tolperisone concentrations of 7.0 mg/l, 14 mg/l and 19 mg/l were found in the blood of the deceased. In the absence of other autopsy findings, the deaths in these three cases were finally explained as a result of lethal tolperisone ingestion. To the best of our knowledge, these three cases are the first reported cases of suicidal tolperisone poisonings.     

Housing instability is as strong a predictor of poor health outcomes as level of danger in an abusive relationship: findings from the SHARE Study

Advocates, clinicians, policy makers, and survivors frequently cite intimate partner violence (IPV) as an immediate cause of or precursor to housing problems. Research has indicated an association between homelessness and IPV, yet few studies examine IPV and housing instability. Housing instability differs from homelessness, in that someone experiencing housing instability may currently have a place to live but faces difficulties with maintaining the residence. We present baseline findings from a longitudinal cohort study of 278 female IPV survivors with housing as a primary concern. Our analysis indicates the greater the number of housing instability risk factors (e.g., eviction notice, problems with landlord, moving multiple times), the more likely the abused woman reported symptoms consistent with PTSD (p < .001), depression (p < .001), reduced quality of life (p < .001), increased work/school absence (OR = 1.28, p < .004), and increased hospital/emergency department use (OR = 1.22, p < .001). These outcomes persist even when controlling for the level of danger in the abusive relationship and for survivors' drug and alcohol use. Importantly, both housing instability and danger level had stronger associations with negative health outcomes than other factors such as age, alcohol, and drug use; both make unique contributions to negative health outcomes and could contribute in different ways. Housing instability is an important and understudied social determinant of health for IPV survivors. These findings begin to address the literature gap on the relationship between housing instability, IPV, and survivors' health, employment, and utilization of medical care services.     

Psychological profile of male and female animal abusers

This study had three purposes: to explore psychological characteristics of animal abusers (criminal thinking styles, empathy, and personality traits), to replicate previously reported results (past illegal actions, bullying behavior), and to examine potential gender differences. The self-reported animal abuser group was 29 college students who reported two or more incidents of animal abuse; controls were 29 college students matched on age and gender. Participants completed self-report measures of criminal thinking, illegal behaviors, bullying, empathy, and the five-factor personality traits. Results indicated animal abusers had more previous criminal behaviors, were more likely to bully, and had the highest scores on the power orientation criminal thinking scale. Abuser by gender interactions were detected; female animal abusers scored significantly higher on several measures of criminal thinking, were found to be more likely to bully, and exhibited lower scores on measures of perspective taking and empathy compared to female controls.