Oral fluid is a versatile matrix that is proving more popular within forensic toxicology. Its use is multifaceted, and is the preferred matrix in therapeutic drug monitoring and roadside testing of drivers suspected to be under the influence of drugs. Benefits including the difficulty of adulteration, the ease and non-invasiveness of sample collection, the range of analytes that can be detected and decent correlations between concentrations in blood or plasma and oral fluid are some of the reasons for its attractiveness to practitioners and forensic toxicologists. Most often, oral fluid is collected using collection devices which can often include a stabilising buffer. When new collection devices are introduced to the market it is important that their applicability to drug testing is investigated to show they are fit for purpose. One of the newest collection devices on the market is the NeoSAL™ collection device from Neogen. This collector was gravimetrically assessed for oral fluid volume collection and drug recovery. Collection volume adequacy of the NeoSAL™ device was compared to two commonly used, commercially available, collection devices: namely the Immunalysis Quantisal™ and the OraSure Intercept® i2™ collection devices. Results showed that the NeoSAL™ device is capable of collecting more than the volume stated by the manufacturer, similar to the Intercept® i2™ which also over-collected, whereas the Quanitsal™ device collected the stated volume. Drug recoveries from the NeoSAL™ collection pad for all drugs investigated in this thesis exceeded 57% (lower recoveries were observed for temazepam and diazepam). Although amphetamine and methamphetamines are not often abused or encountered in forensic samples in Scotland, they are a global problem and effects of abuse can negatively impact a person’s ability to drive by increasing recklessness and risk-taking. Neat and oral fluid collected using the NeoSAL™ device were used to develop and partially validate a method for the quantification of amphetamine, methamphetamine, MDMA, MDA and MDEA using GC-MS. MDEA was also assessed, but did not give acceptable results for accuracy and precision. A short-term autosampler stability study for the four acceptable analytes showed that they were stable on the autosampler for up to 48 hours (~ 19 °C). Opioid and benzodiazepine drugs are two of the most commonly abused drug groups in Scotland. They are often taken synchronously, and the latter is the most commonly prescribed and encountered drug group in Scotland. With the continuation of opioid epidemics and large numbers of people in opioid-treatment programmes, it is beneficial to have a sensitive and selective method that can be used for the simultaneous analysis of these two drug groups. Research has shown that both drug groups are common in drivers, although symptoms of use include loss of coordination, sedation, and drowsiness. An SPE procedure using LC-MS/MS detection was optimised for the extraction for the concurrent analysis of 5 benzodiazepines and 5 opioid drugs. The method was validated according to the guidelines for method validation in forensic toxicology (SWGTOX 2013). The validated method was successfully applied to paired oral fluid and blood samples collected from 16 benzodiazepine users. The NeoSAL™ collection device showed that good recoveries (>57% for all analytes but diazepam and temazepam), and good detection rates for the 10 analytes studied was possible. Oral fluid and blood results showed a good correlation between the analytes detected and in most cases where there was no overlap, it was possible to explain these discrepancies by metabolism, detection windows, low sample volume, and sensitivities of the respective analytical methods used. Gamma-hydroxybutyrate (GHB) is a short-chain fatty acid that is not only endogenous to the mammalian body, but can also be prescribed medicinally and be used as a drug of abuse. A stability study (over 56 days) of GHB in neat oral fluid was carried out as none have been published in the literature. GHB stability is an important factor to assess due to its short detection window in the more traditional matrices blood and urine. A simple protein precipitation extraction procedure was used, and the analytical GC-MS method was adapted from the in-house method for analysis of GHB/beta-hydroxybutyrate (BHB) in blood. The method was partially validated and the stability of GHB was assessed at two concentrations at three temperatures (fridge ~ 4 °C, freezer ~ -21 °C, and room temperature ~ 20 °C). GHB appeared to be stable at all three temperatures for up to 56 days. Endogenous post-mortem blood concentrations of GHB have been widely studied, however debate is still existent regarding cut-off concentrations that should be applied. Problematic interpretation arises from the post-mortem production, and inter- and intra- individual variation of GHB in the human body. 1811 cases between 2010 and 2016, which did not implicate GHB in the cause of death or where GHB was not suspected to have been used, were extracted from the in-house Forensic Medicine and Science (FMS) database. The majority of cases (51%) were deaths related to alcohol abuse. 76% of cases showed GHB concentrations < 30 mg/L, and 94% of all cases had concentrations of less than 50 mg/L. Results also suggest that the use of a preservative may prevent in vitro formation of post-mortem GHB. 112 cases showed GHB concentrations in excess of 50 mg/L with advanced decomposition, therefore suggesting that decomposition changes may increase GHB concentrations. This was the largest dataset that ever studied endogenous post-mortem GHB concentrations, and results highlight the difficulty when applying cut-off concentrations to distinguish post-mortem or exogenous and endogenous concentrations.

Unlike conventional biological samples (blood and urine), hair samples have a much wider detection period and can provide a retrospective timeline of an individual’s drug use. However, the most crucial issue facing hair analysis is the avoidance of false-positive results caused by passive exposure to the drug. Passive exposure could be a result of direct contact with the consumed material or its smoke. This issue is of great concern especially with the drugs that have a greater potential for external contamination. Common examples of these are cannabis and nicotine, two drugs that are by far the most used drugs worldwide. The work presented in this thesis describes the development and validation of three analytical methods for cannabis and nicotine in hair matrices. These methods were then employed to analyse authentic hair samples and their washes. The first method involved liquid-liquid extraction (LLE) of the cannabinoids, Δ9- tetrahydrocannabinol (THC), cannabidiol (CBD), cannabinol (CBN) and metabolite 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC) from hair followed by analysis using standard gas chromatography-mass spectrometry (GC-MS). Cyclohexane: EtOAc (3/1, v/v) was found be the best extracting solvent for THC, CBD, CBN and 11-OH-THC. The percentage of extraction recovery for all four analytes ranged from 87.9% to 97.2%. The second method involved solid-phase extraction (SPE) of the main metabolite 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) from hair followed by analysis using two-dimensional gas chromatography-mass spectrometry (2D GC-MS). The SPE method provided a clean extract with an acceptable extraction recovery (approximately 50%). Authentic hair samples were then collected from 20 known cannabis users admitted to Al-Amal addiction hospital in Jeddah, Saudi Arabia. Cannabis users were interviewed at the time of sample collection and self-reported their cannabis use history. Concentrations of different cannabinoids were then measured using the validated methods. The aim of this project was to investigate the potential value of measuring cannabinoid concentrations in hair. The detected concentrations ranged from 0.11 to 0.34 ng/mg for THC, 0.2 to 4.42 ng/mg for 3 CBD, 0.31 to 1.02 for CBN, and 2.14 to 7.01 pg/mg for THC-COOH. Surprisingly, THC has a very low detection rate, whereas, CBD and THC-COOH had the highest detection rate of all cannabinoids. The relationship between measured concentrations and use history was then subject to statistical analysis. There was no significant correlation found between concentrations of cannabinoids in hair and the use history. The third method involved methanolic extraction of nicotine and cotinine from pet dogs’ fur followed by analysis by zwitterionic hydrophilic interaction liquid chromatography tandem mass spectrometry (ZICHILIC-MSMS). Further clean-up of the fur methanolic extract was found to be problematic. Centrifugation and direct analysis was found to be the best approach. The tandem MS allowed for low detection limits. The aim of this project was to investigate the association between dog fur nicotine and cotinine concentrations and owner-reported exposure to environmental tobacco smoke. 66 fur samples were collected from 41 dogs at two time points. Total nicotine and total cotinine were quantified in unwashed fur samples using the validated method. Statistical analysis revealed a significant difference in the mean concentrations of nicotine and cotinine in different exposure groups. By providing information on dog’s exposure to environmental tobacco smoke (ETS) over time, fur analysis may be useful in assessing dogs and companion owner’s histories of exposure to ETS.

Orientador: Claudio José Von Zuben / Banca: Arício Xavier Linhares / Banca: Wesley Augusto Conde Godoy / Banca: Janyra Oliveira Costa / Banca: Margareth M.M. Queiroz / Resumo: Um dos principais propósitos da investigação de fenômenos em qualquer área de atuação é encontrar uma forma adequada de representação dos processos envolvidos que seja o mais simples possível. O estudo da auto-organização em agregados de organismos biológicos não pode fugir a este propósito, e foi realizado aqui em três etapas: experimentação, modelagem e validação. A experimentação vai permitir definir claramente os padrões de comportamento a serem analisados e interpretados. A modelagem irá recorrer a estratégias espaço-temporais para a síntese de explanações matemáticas, para as interações e modos de agir dos organismos. Finalmente, a validação estará baseada em simulações computacionais, comparações com os resultados experimentais e interpretação paramétrica dos modelos resultantes. Foi predominante junto ao processo de modelagem o uso apenas das informações provenientes das observações realizadas e dados experimentais coletados, caracterizando uma abordagem de modelagem bottom-up, em detrimento da incorporação de hipóteses intuitivas a respeito dos mecanismos envolvidos, procedimento típico das abordagens top-down. Além disso, a acurácia do modelo em reproduzir resultados experimentais não foi o único e principal fator de avaliação da qualidade dos modelos resultantes, pois o aspecto mais importante a ser considerado junto aos modelos é a capacidade de ampliar o conhecimento acerca do fenômeno envolvido. Embora existam muitas manifestações de auto-organização na natureza e que poderiam ser tomadas como casos de estudo, este projeto se propôs a investigar o processo de dispersão larval pós-alimentar em moscas-varejeiras do gênero Chrysomya. Essas moscas foram introduzidas no Brasil em meados dos anos 70, e são de considerável importância médico-sanitária... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: In any area of investigation, one of the main goals is to identify the simplest and most adequate representation of the phenomenon being studied. The study of selforganization in aggregates of organisms is no exception to this rule. In the work described here, we used a combination of laboratory experiments, modeling and validation to clearly identify patterns of behavior. For modeling, spatiotemporal strategies were used to develop mathematical explanations of the interactions and ways organisms act. The final validation was based on computer simulations, comparisons with experimental results and parametric interpretation of the resulting models. The modeling was based on observations and data collected during the experiments, an approach known as bottom-up modeling, and which contrasts with top-down modeling that depends on the incorporation of intuitive hypotheses about the mechanisms involved. Apart from the accuracy of the model in reproducing the experimental results, the most important aspect to be considered is the ability of the resulting model(s) to increase our knowledge about a given phenomenon. In this investigation, we used postfeeding larval dispersal in blowflies of the genus Chrysomya as a model to study self-organization. Chrysomya blowflies were introduced into Brazil in the mid-1970s, and are of considerable medicosanitary importance since they are vectors of enteropathogens that cause facultative myiasis in animals and man; these flies are also used in forensic studies to estimate the postmortem interval (PMI) in humans. The larval stage in these flies is the principal period in which there is competition for discrete, ephemeral food resources in the wild. After exhaustion of the food resources, the larvae leave the food... (Complete abstract, click electronic address below) / Doutor

A língua humana foi primeiramente descrita por Andreas Vesalius, o &ldquo;Pai da Anatomia Moderna&rdquo;, no livro De humani corporis Fabrica em 1543. Contudo, as papilas do dorso da língua eram desconhecidas até então. Estas foram descritas e classificadas nos seus três tipos básicos de acordo com o tamanho em papilas de 1&ordf;, 2&ordf; e 3&ordf; ordens somente após o advento do microscópio, por Marcello Malpighi em 1665. A partir daí, os padrões morfológicos das papilas variaram de acordo com a descrição de diferentes autores até alcançar os quatro tipos conhecidos até os dias de hoje, sendo elas: fungiformes, filiformes, folhadas e circunvaladas, sendo estas últimas o objeto de estudo do presente trabalho. Sugeridas primeiramente como tema de investigação para Alfonso Bovero (1871- 1937) fundador da Escola Anatômica de São Paulo pelo seu mentor Carlo Giacomini (1840-1898), Bovero criou um acervo na Faculdade de Medicina da Universidade de São Paulo (FM-USP) com mais de 800 línguas humanas obtidas de indivíduos masculinos e femininos, de diferentes idades e etnias. Em seu trabalho preliminar sobre o tema, publicado em 1936, ele descreveu o elevado grau de polimorfismo das papilas, agrupando-as a outros &ldquo;instrumentos de função&rdquo; como o encéfalo e as mãos humanas que, como órgãos da vida de relação, se caracterizam pela extrema complexidade morfológica. Com a sua morte em 1937, a pesquisa foi interrompida e do acervo inicial restaram 327 espécimes. O presente trabalho resgatou esse material a fim de avaliar com metodologias atuais a morfologia das papilas, levando em consideração a hipótese de individualidade lingual postulada inicialmente por Bovero, de que não existem duas línguas idênticas, nem mesmo nos antímeros de uma mesma língua. Para identificar os padrões estruturais e de possível singularidade das papilas disposição, número, área papilar, presença ou ausência de orla e formas tanto da própria papila quanto da sua orla foram utilizadas as técnicas de Morfometria, Mesoscopia, e Microscopias de Luz e Eletrônica de Varredura. Os resultados demonstram que no geral as línguas estão sobre 4 formas (circuliformes, trianguliformes, fusiformes e retanguliformes), as PCVs estão dispostas principalmente em forma de &rdquo;V&rdquo; e &ldquo;Y&rdquo; linguais; elas podem ser verdadeiras ou falsas; algumas papilas apresentaram a mesma área, porém com morfologia diferente. Além disso, houve variação na textura dos componentes das PCVs. A combinação das variáveis estudadas (papilas, orlas, valo, poros gustatórios e histologia) confirmam a hipótese de unicidade glótica. Dessa forma, os remanescentes do acervo com um século de existência foram aproveitados, demonstrando viabilidade para o estudo anatômico e valorizando os experimentos iniciais do fundador da Escola Boveriana de Anatomia / The human tongue was first described by Andreas Vesalius, known as &ldquo;the father of modern anatomy&rdquo;, in the book De humani corporis Fabrica, published in 1543. However, at that time, the dorsal lingual papillae were still unknown. They were described and classified according to size in three basic types: 1st, 2nd and 3rd order papillae, only after the advent of the microscope, by Marcello Malpighi in 1665. Thereafter, the morphological patterns of papillae varied according to different authors&#39; descriptions until reaching the four currently known types: fungiform, filiform, foliate and circunvallate. The later is the aim of this work. Influenced by his mentor, Carlo Giacomini (1840-1898), Alfonso Bovero (1871- 1937) founder of the School of Anatomy of São Paulo created an archive of over 800 human tongues, from male and female individuals, at different ages and different ethnicities at the School of Medicine of the University of São Paulo. In his early work on this subject, published in 1936, he described the high degree of polymorphism found among the papillae, classifying them as other &quot;function instruments&quot;, such as the encephalon and the human hands, which, as organs of relation, are characterized by their high morphological complexity. With his death in 1937, this study was interrupted and currently only 327 specimens are left from the original archive. This work analyzed this material regarding papillae morphology with currently available technologies, considering the lingual individuality hypothesis initially proposed by Bovero, which states that there are no two identical tongues or antimers on a given tongue. To identify the papillae&#39;s structural and possible singularity patterns distribution, number, papillary area, presence or absence of rims, and shape of both the papillae and their rims we used morphometry, mesoscopy, and light and scanning electronic microscopy. Results show that, in general, tongues are presented in one of four shapes (circle, triangle, rectangles, and fusiforms); vallate papillae (PCVs) are distributed on the tongue mainly as V- or Y-shaped regions; they can be true or false; some papillae have the same area, but different morphologies. In addition to that, we found variation on the texture of PCVs components. In combination, the studied variables (papillae, rims, pits, taste pores, and hystology) corroborate the hypothesis of uniqueness of the tongue. Thus, the remaining of this one-century-old archive were used, viability for the anatomical study was showed, and the initial experiments of the Boverian School of Anatomy founder were praised

New psychoactive substances (NPSs) have appeared on the recreational drug market at an unprecedented rate in recent years. Many are not new drugs but failed products of the pharmaceutical industry. The speed and variety of drugs entering the market poses a new complex challenge for the forensic toxicology community. The detection of these substances in biological matrices can be difficult as the exact compounds of interest may not be known. Many NPS are sold under the same brand name and therefore users themselves may not know what substances they have ingested. The majority of analytical methods for the detection of NPSs tend to focus on a specific class of compounds rather than a wide variety. In response to this, a robust and sensitive method was developed for the analysis of various NPS by solid phase extraction (SPE) with gas chromatography mass spectrometry (GCMS). Sample preparation and derivatisation were optimised testing a range of SPE cartridges and derivatising agents, as well as derivatisation incubation time and temperature. The final gas chromatography mass spectrometry method was validated in accordance with SWGTOX 2013 guidelines over a wide concentration range for both blood and urine for 23 and 25 analytes respectively. This included the validation of 8 NBOMe compounds in blood and 10 NBOMe compounds in urine. This GC-MS method was then applied to 8 authentic samples with concentrations compared to those originally identified by NMS laboratories. The rapid influx of NPSs has resulted in the re-analysis of samples and thus, the stability of these substances is crucial information. The stability of mephedrone was investigated, examining the effect that storage temperatures and preservatives had on analyte stability daily for 1 week and then weekly for 10 weeks. Several laboratories identified NPSs use through the cross-reactivity of these substances with existing screening protocols such as ELISA. The application of Immunalysis ketamine, methamphetamine and amphetamine ELISA kits for the detection of NPS was evaluated. The aim of this work was to determine if any cross-reactivity from NPS substances was observed, and to determine whether these existing kits would identify NPS use within biological samples. The cross- reactivity of methoxetamine, 3-MeO-PCE and 3-MeO-PCP for different commercially point of care test (POCT) was also assessed for urine. One of the newest groups of compounds to appear on the NPS market is the NBOMe series. These drugs pose a serious threat to public health due to their high potency, with fatalities already reported in the literature. These compounds are falsely marketed as LSD which increases the chance of adverse effects due to the potency differences between these 2 substances. A liquid chromatography tandem mass spectrometry (LC-MS/MS) method was validated in accordance with SWGTOX 2013 guidelines for the detection for 25B, 25C and 25I-NBOMe in urine and hair. Long-Evans rats were administered 25B-, 25C- and 25I-NBOMe at doses ranging from 30-300 µg/kg over a period of 10 days. Tail flick tests were then carried out on the rats in order to determine whether any analgesic effects were observed as a result of dosing. Rats were also shaved prior to their first dose and reshaved after the 10-day period. Hair was separated by colour (black and white) and analysed using the validated LC-MS/MS method, assessing the impact hair colour has on the incorporation of these drugs. Urine was collected from the rats, analysed using the validated LC-MS/MS method and screened for potential metabolites using both LC-MS/MS and quadrupole time of flight (QToF) instrumentation.

The work described in this thesis deals with three aspects of quality assurance in the field of forensic toxicology: proficiency testing schemes, validation of analytical methods for the piperazine group of abused drugs and validation of the police field impairment test, used at the roadside to test drivers for drug-induced impairment. Proficiency Testing: Long term reviews were performed for two forensic external quality assurance schemes. Rounds 30 (in 2007) to 48 (in 2012) of the UKAS-accredited commercial Quartz Forensic Blood Toxicology Proficiency Testing Scheme (PTS), and a ten year period from 1999 to 2009 of the freely-available United Nations Office on Drugs and Crime (UNODC) International Collaborative Exercises (ICE). Only limited ICE data could be made available as much of the original data had been stored on a database which had become obsolete, hence the data were only available as the original results forms provided to UNODC by the ICE participants. Data was entered to Microsoft Excel® spreadsheets and Microsoft Access® databases from the original forms for the years 1999, 2001 (2 rounds), 2003 (2 rounds) and 2005 (2 rounds), and summary data was extracted from the UNODC round reports for the years 2007, 2008 and 2009. Four methods of scoring quantitative performance were reviewed and the most suitable, a z-score using an assigned ‘true’ value and a percentage of the true value as acceptable deviation, was applied to reanalyse the participants’ results and assess their performance. Methods of scoring proficiency which relied upon participants’ data to determine acceptable variation were found merely to describe the data rather than challenge participants on whether or not they were performing fit-for-purpose analyses. Factors such as participation, analytes tested, participants’ methods of analysis and participants performance were summarised for each scheme before the performance of the two schemes, and that of their participants, were compared. ICE tested more analytes per annum but from a smaller test menu than Quartz. This resulted in more repetitive testing and allowed for some trend analysis and performance monitoring. It was not possible to observe performance trends with Quartz due to the wide variety of analytes tested. The smaller array of potential analytes and more repetitive nature of ICE testing also meant that performance monitoring and detection of bias were easier to perform, and ICE was shown to be more effective as external quality assurance (EQA). Quartz provided a good educational resource as it incorporated the wide range of drugs which a forensic toxicology laboratory could realistically encounter. Following the review, however, it was recommended for QUARTZ that, to provide a safeguard against bias, more repetitive testing was required and this has now been adopted. Piperazines: All piperazine analogues are now illegal in the UK, registered as Class C of the Misuse of Drugs Act (1971) and schedule 2, part III of the Misuse of Drugs Regulations (2001). Piperazines can elicit similar effects to some ATS and methods for their detection should be available in forensic toxicology laboratories. In the present study, methods were developed for the detection of a range of piperazines in blood using LC-MS/MS (p-MeOPP, p-FPP, BZP, o-MeOPP, p-MPP and TFMPP) and GC-MS (p-FPP, BZP, TFMPP, p-MPP, o-MeOPP, m-CPP, p-MeOPP and p-CPP). Quality assurance required both methods to be validated. For all piperazine analytes accuracy was within ±15% (20% at low concentrations) and precision was within 15% (20% at low concentrations). For both methods LLOD of all analytes was 5 ng/ml of blood and upper limit of quantification was 2 µg/ml of blood. For the GC-MS method lower limits of quantification (LLOQs) were in the range 20 to 30 ng/ml of blood. For LC-MS/MS, LLOQs ranged from 50 to 60 ng/ml of blood, although quantification by the LC-MS/MS method was restricted by the lack of availability of appropriate internal standards. There were no apparent significant matrix effects and recovery by both methods was >60 % and, therefore, acceptable. Short term stability of the piperazine analytes was investigated. Piperazines remain sufficiently stable when stored in the fridge for at least one week, and are stable through three freeze-thaw cycles. There was no detectable degradation when blood samples were left on the bench-top or when extracted ‘in-process’ samples were left in the autosampler for up to 72 hours. The LC-MS/MS method could provide a readily applicable screening method. A small aliquot of a basic drug extract could be screened by LC-MS/MS for the presence of piperazines, leaving the majority of the extract for other analyses, for example, piperazines confirmation or amphetamines analysis. The GC-MS method was suitably validated to provide quantification but application to casework samples remains to be evaluated. It is recommended that piperazine testing be performed for all suspected MDMA or ‘club drug’ intoxication cases. The Field Impairment Test: The detection of drugged drivers primarily depends on the current method which is the driver field impairment test (FIT). FIT comprises measurement of pupil diameter and four physical tasks (the Romberg balance test, walk and turn test, one legged stand and finger to nose test) intended to simultaneously test comprehension, short term memory, balance and motor function. Despite FIT having ISO accreditation, it has been recognised that police officers lack confidence with the protocol and do not apply the test as often as is necessary. The main difficulty arises from the requirement to make a subjective judgement of impairment and officers lack confidence in their ability to do so. FIT has never been fully validated. The present study was designed to meet the urgent requirement to develop FIT into an objective measurement, by determining what constitutes “normal” performance in FIT by unimpaired adults of different ages. FIT performance was recorded for 79 individuals, a statistically determined cohort size, confirmed by breath and oral fluid analysis not to be under the influence of impairing substances. Each error made during FIT, as defined by the FIT standard operating procedure, was recorded and collated in a Microsoft Excel® spreadsheet for analysis. It was found that the definition of ‘errors’ was too stringent as many which are required to be recorded are normal physiological or behavioural characteristics, such as body sway, and most subjects would be unable to complete the task without displaying them. A less stringent, evidence-based definition of “error” was developed which allowed statistically more significant analysis to be performed on the FIT results. A statistically significant difference (P=0.00578) was shown to exist between the FIT performance of individuals under the age of forty years and those aged forty and over. Based on the principles of a PTS, robust mean and standard deviation were used to determine what constituted acceptable performance. Those in the younger age group could be considered impaired if the police officer witnessed more than seven errors, or, in the older age group, more than fifteen errors. Using these criteria the frequency of false positives, i.e. unimpaired drivers being assessed as impaired is estimated to be (less than 3%). Also, the ranges of errors observed in both groups was large and overlapped, such that it may be possible for an impaired person to appear unimpaired. This requires further investigation.

This research was conducted to provide empirical evidence to supplement advice available to the forensic community for the collection of muscle tissue for forensic analysis. This type of collection is normally carried out to determine the identity of individuals following mass disasters, such as plane crashes or natural disasters. DNA degradation was assessed in two model organisms, pig and rabbit (with human DNA as a control), over various time points. Rabbit recombination activating gene (RAG 1) was aligned to identify conserved regions in pig, rabbit and human. Primers were designed and optimised to create a 4-plex PCR multiplex that can amplify 70 bp, 194 bp, 305 bp and 384 bp in three species. The 4-plex multiplex was found to work efficiently in all three species down to 0.3 ng of DNA template. The multiplex was used to assess whether DNA degradation can be predicted by accumulated degree-days (ADD), which provides a measure of both time and temperature. A series of field studies were performed to assess DNA persistence in pig and rabbit soft muscle tissues using a combination of whole animals, suspended muscle tissues (insect activity free) and muscle fragments. Field studies were carried out in: August-September 2009; February-May 2010; May-June 2010; June-July 2010 and September-November 2010. Soft muscle tissue samples were collected at different ADD. 4-plex multiplex results showed that DNA was more persistent in pig tissues compared to rabbit tissues. In the September 2010 experiments, full multiplex amplification was obtained from rabbit until 137 ADD (whole carcases) and 210 ADD (body fragments and suspended tissues), while in the August 2009 experiments, full multiplex amplification was obtained until 112 ADD (whole carcases and body fragments) and until 141 ADD (suspended tissues). In the June 2010 experiments, full multiplex amplification was possible until 64 ADD. Pig whole carcases which were placed in the field in February 2010, showed multiplex amplification until day 90 (603 ADD), followed by September 2010 (until day 44 (490 ADD)) and May 2010 (until day 27 (338 ADD)). During the September 2010 project, body fragments produced full amplification until muscles were collected (342 ADD), while in case of whole carcases and suspended tissues; the amplification was possible until 490 ADD. There was complete failure of amplification of 305 bp and 384 bp in pig whole carcases after 342 ADD, while in suspended tissues, the amplification of 305 bp and 384 bp was possible until 420 ADD. The statistical analysis showed that amplification success of larger amplicons (194 bp, 305 bp and 384 bp) reduces with increase in ADD in pig and rabbit whole carcases, body fragments and suspended tissues while 70 bp was more persistence. The results showed that there was no significant difference in DNA persistence between whole carcases verses suspended tissues (Z=0.57, p>0.05) and whole carcases verses body fragments (Z=1.71, p>0.05), There was however a significant difference (Z=2.31, p<0.05) in DNA persistence in suspended tissues and body fragments with increase in ADD. The results from field experiments suggested that muscle tissues, if available, should be collected for DNA profiling, since even if degraded, a profile can be obtained. The results also suggested that the isolation of tissues from insect activity as quickly as possible (even if immediate storage is not possible) may be beneficial for DNA persistence. Seasonal variation in DNA persistence was observed due to maggot mass growth which increases carcase decomposition and ultimately effect on DNA persistence. Controlled incubation experiments were also performed at 27 °C, 37 °C and 47 °C until 21 days to assess DNA persistence, as these temperatures were not available under field conditions. The results showed that the amplification of 70 bp was more persistent compared to larger amplicons (194 bp, 305 bp and 384 bp). The drop-out in amplification of larger amplicons occurred more rapidly in samples incubated under laboratory conditions compared to the field samples. The statistical analysis showed species, ADD and temperature have strong effect (p<0.05) on DNA persistence under controlled conditions. The appearance of 70 bp amplicons in all samples collected from field and in most samples from controlled incubation experiments suggested that soft muscle tissues exposed to different environments can be used to perform SNP analysis. The full 4-plex multiplex amplification obtained from rabbit and pig preserved and dehydrated samples suggested that 96% ethanol, cell lysis solution (with and without 1% sodium azide) and dehydration can be used to preserve fresh and partially decomposed soft muscle tissues at room temperature for one year. The drop-out in amplification of larger amplicons in tissues preserved in 10% buffered formalin suggested that formalin was not suitable for long term storage. This system should therefore be considered as an additional method during Disaster victim identification (DVI) work to preserve fresh and partially decomposed samples. This study also suggested that the developed multiplex (4-plex) can be used to assess DNA persistence in human decomposing bodies and in experimental studies.

Single nucleotide polymorphisms (SNPs) are one of the forensic markers used to resolve the problem of DNA typing from degraded samples. It has been found in previous studies that when profiling heavily degraded forensic samples the small amplicon required for SNP analysis has an advantage over the larger STR loci, which are routinely used in forensic case work. A total of 66 SNPs from the non-coding region of the 22 pairs of autosomal chromosomes were identified and SNP assays developed. Instead of selecting the SNPs from the available GenBank® sites, SNPs were typed from Arab individuals from Kuwait and United Arab Emirates (UAE) to identify polymorphic SNPs. In order to obtain SNP data from Arab populations, a total of 10 unrelated Arab individuals from Kuwait and UAE were typed. The Affymetrix GeneChip® Mapping 250 K Array Sty І was employed to generate profiles for approximately 238,000 SNPs. Only autosomal SNPs were selected from the data. Following selection, allele frequencies were estimated using the SNaPshot™ technique (Applied Biosystems) with 25 UAE individuals. For this technique, PCR forward and reverse primers were designed to generate PCR products less than 150 bp. The single base extension primers were designed to hybridise 1 bp upstream from the target SNP. SNP characterization, including Hardy¬Weinberg equilibrium and pair wise linkage disequilibrium, was carried out using the software package Arlequin v 3.1. Allele frequencies were calculated using Excel spreadsheets. PowerStats v.12 software used for discrimination power and match probability estimation. All the 66 SNPs were polymorphic with average heterozygosity levels of 47%. A high heterozygosity level is very valuable for forensic application improving the individualization of forensic samples (Vallone et al. 2005). The probability that two individuals having identical genotype profile was found to be very low, 3.058 x 10-25. The combined power of discrimination was found to be 0.999999999. This indicated that the selected SNPs met the parameters needed for forensic application. The SNPs genotype sensitivity gave profiles from minute amounts of DNA template as little as 100 pico grams (pg) and optimal and reproducible results at 300 pg of DNA template. The profiling of DNA from forensic samples is not always possible. This can be due to insufficient amount of samples being recovered and in many cases, DNA degradation. Biological materials that are recovered from the scene of the crime have often been exposed to sub-optimal environmental conditions such as high temperature and humidity. SNPs performance on degraded samples was tested on artificially degraded saliva and semen samples. Controlled temperature and humidity experiments were performed to study the effect of these environmental factors on the samples. Also uncontrolled experiments on samples being subjected to different weather conditions (UK summer and UAE winter and summer) was performed in order to study and compare both weather effects on saliva samples. The triplex sets of SNPs that were developed for such study showed full allele profiles when compared to STRs, the current method used in forensic labs. In addition, SNPs produced a higher success rate than STRs when tested with samples obtained from human teeth remains and on samples subjected to DNase 1 digestion. The small size of SNPs, between 90 and 147 base pair (bp), showed more resistance to degradation than the STRs size ranging between 100 and 360 bp. This study demonstrated that the 66 SNPs selected are useful markers when the typing of degraded samples by STRs fails to produce complete or partial profiles.

Cut mark analysis to date has been intermittently and superficially researched across a range of disciplines, despite its potential to significantly contribute to criminal investigation. The current study aims to elucidate cut mark analysis by proposing a novel classification system for the identification of knife cuts (kerfs) in bone. The system was devised, to record accurate and reliable information about cut marks and the criteria were tested for association with the knives that created them. Optical Microscopy was used to examine knife cuts on fleshed porcine bone. Incised cuts were made by a range of serrated, scalloped and fine-edged blades (n=9), by the author, and participants (n=23) were recruited to make marks on bone under the same force-measured conditions, using the Kistler force plate and a bespoke frame to control the level of height to which the knife can be raised above the bone prior to impact. Resultant kerfs were created by a single operator (n=86) and created by a range of individuals (n=186). The data suggests that consistent force was not achieved and the resultant marks on the bones made by the same knife had wide variation in their appearance and depth. The classification criteria tested did not provide discrete identification of knife blades from the assessment of kerf features; however, trends were identified from criteria including margin regularity, margin definition, floor width and wall gradient, which may form the basis for further investigation. Marks made by a single operator showed more significant associations (p<0.05) than group operators, and although kerfs from each share some trends, several significant relationships observed in marks made by a single operator are not shared by the participant group. Limitations of using optical microscopy included the inability to view all aspects of each mark, particularly when combined with variation in depth and angle produced by human operators. From the present results, it is suggested that the use of digital microscopy with a superior ability to build three dimensional images of indented marks would provide the necessary step forward to improve discrimination between knife classifications, based on the areas highlighted by the current research. This reinforces the need for further understanding of the mechanics of cut mark application in human individuals and their potential effects on kerf features.

Three different derivatisation procedures were evaluated for their general applicability to systematic toxicological analysis (STA) involving (a) acylation with pentafluoropropionyl anhydride (PFPA) and pentafluoropropanol (PFP-OH), (b) acylation/esterification (methylation) with pentafluoropropionyl anhydride (PFPA) and a novel methylating agent trimethylsilyldiazomethane (TMS-diazomethane), used as a chromatographic derivatisation reagent for the first time in this study, and (c) silylation with tertiary-butyldimethylsilyl-trifluoroacetamide (MTBSTFA). Model compounds were selected for the evaluation process including a primary amine (amphetamine), a secondary amine (methamphetamine), alicyclic and aromatic hydroxy compounds (morphine, tetrahydrocannabinol), and carboxylic acids (benzoylecgonine, 11-nor tetrahydrocannabinol-9-carboxylic acid). For method (a) derivatisation was successful for all of the test compounds and mass spectra were obtained for each of them. For method (b), the novel methylating agent trimethylsilyl-diazomethane was used to convert carboxylic acids into the corresponding methyl esters. This reaction was found to proceed rapidly and quantitatively at room temperature and holds potential for future use in toxicology to replace diazomethane, a hazardous and toxic material. Method (c) gave derivatives with all test compounds except the secondary amine, methamphetamine, and the alcohol, morphine. The gas chromatographic behaviour of these derivatives was good and the mass spectra had prominent ions suitable for GC-MS-SIM. The extraction of multiple drugs from blood was evaluated using the novel polymeric SPE sorbent Strata-X. The same test compounds were used to assess the extraction step in terms of recovery and variation (within day and between days). The extracts were analysed by GC-MS-SIM using each of the three types of derivative. Recoveries of the test compounds were in the range of 50-100 percent depending on the analyte and its concentration in blood. All calibration curves were linear and had correlation coefficients higher than 0.99. Within day variations and between day variations were in the range of 2-22% relative standard deviation. Limits of detection and quantitation were measured for the model compounds and were found to be in the ranges of 0.4-7.3 ng/ml and 1.1-24.4 ng/ml respectively. The full method, combining extraction with each of the derivatisation reactions was finally evaluated for the presence of interferences with real case blood samples. The three derivatisation procedures were evaluated using four test compounds comprising diazepam plus its three metabolites nordiazepam, temazepam and oxazepam. The hydroxylated metabolites (temazepam and oxazepam) formed derivatives readily with all three reagent mixtures but nordiazepam (secondary aromatic amine) did not react except with MTBSTFA. Based on the work of this study it is concluded that a method is possible for STA based on a polymeric sorbent, to give a general extract, followed by a generalised derivatisation procedure such as acylation, with PFPA/PFP-OH prior to GC-MS.