In vitro studies of drug transformations: application to forensic toxicology

The forensic toxicologist faces challenges in the detection of drugs and poisons in biological samples due to transformations which occur both during life and after death. For example, changes can result from drug metabolism during life or from the use of formalin solution for post mortem embalming purposes. The former requires the identification of drug metabolites and the latter the identification of chemical reaction products in order to know which substances had been administered. The work described in this thesis was aimed at providing ways of tackling these challenges and was divided into two parts. Part 1 investigated the use of in vitro drug metabolism by human liver microsomes (HLM) to obtain information on drug metabolites and Part 2 investigated the chemical reactions of drugs and a carbamate pesticide with formalin solution and formalin-blood. The initial aim of part I was to develop an in vitro metabolism method using HLM, based on a literature review of previous studies of this type. MDMA was chosen as a model compound to develop the HLM method because its metabolism was known and standards of its metabolites were commercially available. In addition, a sensitive and selective method was developed for the identification and quantitation of hydrophilic phase I drug metabolites using LC/MS/MS with a conventional reverse-phase (C18) column. In order to obtain suitable retention factors for polar drug metabolites on this column, acetyl derivatives were evaluated for converting the metabolites to more lipophilic compounds and an optimal separation system was developed. Acetate derivatives were found to be stable in the HPLC mobile phase and to provide good chromatographic separation of the target analytes. In vitro metabolism of MDMA and, subsequently, of other drugs involved incubation of 4 µg drug substance in pH 7.4 buffer with an NADPH generating system (NGS) at 37oC for 90 min with addition of more NGS after 30 min. The reaction was stopped at 90 min by the addition of acetonitrile before extraction of the metabolites. Acetate derivatives of MDMA metabolites were identified by LC/MS/MS using multiple reaction monitoring (MRM). Three phase I metabolites (both major and minor metabolites) of MDMA were detected in HLM samples. 3,4-dihydroxy-methamphetamine and 4-hydroxy-3-methoxymethamphetamine were found to be major metabolites of MDMA whereas 3,4-methylenedioxyamphetamine was found to be a minor metabolite. Subsequently, ten MDMA positive urines were analysed to compare the metabolite patterns with those produced by HLM. An LC/MS method for MDMA and its metabolites in urine samples was developed and validated. The method demonstrated good linearity, accuracy and precision and insignificant matrix effects, with limits of quantitation of 0.025 µg/ml. Moreover, derivatives of MDMA and its metabolites were quantified in all 10 positive human urine samples. The urine metabolite pattern was found to be similar to that from HLM. The second aim of Part 1 was to use the HLM system to study the metabolism of some new psychoactive substances, whose misuse worldwide has necessitated the development of analytical methods for these drugs in biological specimens. Methylone and butylone were selected as representative cathinones and para-methoxyamphetamine (PMA) was chosen as a representative ring-substituted amphetamine, because of the involvement of these drugs in recent drug-related deaths, because of a relative lack of information on their metabolism, and because reference standards of their metabolites were not commercially available. An LC/MS/MS method for the analysis of methylone, butylone, PMA and their metabolites was developed. Three phase I metabolites of methylone and butylone were detected in HLM samples. Ketone reduction to β-OH metabolites and demethylenation to dihydroxy-metabolites were found to be major phase I metabolic pathways of butylone and methylone whereas N-demethylation to nor-methylone and nor-butylone were found to be minor pathways. Also, demethylation to para-hydroxyamphetamine was found to be a major phase I metabolic pathway of PMA whereas β-hydroxylation to β-OH-PMA was found to be a minor pathway. Formaldehyde is used for embalming, to reduce decomposition and preserve cadavers, especially in tropical countries such as Thailand. Drugs present in the body can be exposed to formaldehyde resulting in decreasing concentrations of the original compounds and production of new substances. The aim of part II of the study was to evaluate the in vitro reactions of formaldehyde with selected drug groups including amphetamines (amphetamine, methamphetamine and MDMA), benzodiazepines (alprazolam and diazepam), opiates (morphine, hydromorphone, codeine and hydrocodone) and with a carbamate insecticide (carbosulfan). The study would identify degradation products to serve as markers for the parent compounds when these were no longer detectable. Drugs standards were spiked in 10% formalin solution and 10% formalin blood. Water and whole blood without formalin were used for controls. Samples were analysed by LC/MS/MS at different times from the start, over periods of up to 30 days. Amphetamine, methamphetamine and MDMA were found to rapidly convert to methamphetamine, DMA and MDDMA respectively, in both formalin solution and formalin blood, confirming the Eschweiler-Clarke reaction between amine-containing compounds and formaldehyde. Alprazolam was found to be unstable whereas diazepam was found to be stable in both formalin solution and water. Both were found to hydrolyse in formalin solution and to give open-ring alprazolam and open-ring diazepam. Other alprazolam conversion products attached to paraformaldehyde were detected in both formalin solution and formalin blood. Morphine and codeine were found to be more stable than hydromorphone and hydrocodone in formalin solution. Conversion products of hydromorphone and hydrocodone attached to paraformaldehyde were tentatively identified in formalin solution. Moreover, hydrocodone and hydromorphone rapidly decreased within 24 h in formalin blood and could not be detected after 7 days. Carbosulfan was found to be unstable in formalin solution and was rapidly hydrolysed within 24 h, whereas in water it was stable up to 48 h. Carbofuran was the major degradation product, plus smaller amounts of other products, 3-ketocarbofuran and 3-hydrocarbofuran. By contrast, carbosulfan slowly hydrolysed in formalin-blood and was still detected after 15 days. It was concluded that HLM provide a useful tool for human drug metabolism studies when ethical considerations preclude their controlled administration to humans. The use of chemical derivatisation for hydrophilic compounds such as polar drug metabolites for analysis by LC/MS/MS with a conventional C18 column is effective and inexpensive, and suitable for routine use in the identification and quantitation of drugs and their metabolites. The detection of parent drugs and their metabolites or conversion and decomposition products is potentially very useful for the interpretation of cases in forensic toxicology, especially when the original compounds cannot be observed.

Managing university records and documents in the world of governance, audit and risk: Case studies from South Africa and Malawi

There are enormous benefits for any organisation from practising sound records management. In the context of a public university, the importance of good records management includes: facilitating the achievement the university’s mandate; enhancing efficiency of the university; maintaining a reliable institutional memory; promoting trust; responding to an audit culture; enhancing university competitiveness; supporting the university’s fiduciary duty; demonstrating transparency and accountability; and fighting corruption. Records scholars and commentators posit that effective recordkeeping is an essential underpinning of good governance. Although there is a portrayal of positive correlation, recordkeeping struggles to get the same attention as that given to the governance. Evidence abounds of cases of neglect of recordkeeping in universities and other institutions in Sub-Saharan Africa. The apparent absence of sound recordkeeping provided a rationale for revisiting some universities in South Africa and Malawi in order to critically explore the place of recordkeeping in an organisation’s strategy in order to develop an alternative framework for managing records and documents in an era where good governance is a global agenda. The research is a collective case study in which multiple cases are used to critically explore the relationship between recordkeeping and governance. As qualitative research that belongs in the interpretive tradition of enquiry, it is not meant to suggest prescriptive solutions to general recordkeeping problems but rather to provide an understanding of the challenges and opportunities that arise in managing records and documents in the world of governance, audit and risk. That is: what goes on in the workplace; what are the problems; and what alternative approaches might address any existing problem situations. Research findings show that some institutions are making good use of their governance structures and other drivers for recordkeeping to put in place sound recordkeeping systems. Key governance structures and other drivers for recordkeeping identified include: laws and regulations; governing bodies; audit; risk; technology; reforms; and workplace culture. Other institutions are not managing their records and documents well despite efforts to improve their governance systems. They lack recordkeeping capacity. Areas that determine recordkeeping capacity include: availability of records management policy; capacity for digital records; availability of a records management unit; senior management support; level of education and training of records management staff; and systems and procedures for storage, retrieval and dispositions of records. Although this research reveals that the overall recordkeeping in the selected countries has slightly improved compared with the situation other researchers found a decade ago, it remains unsatisfactory and disjointed from governance. The study therefore proposes governance recordkeeping as an approach to managing records and documents in the world of governance, audit and risk. The governance recordkeeping viewpoint considers recordkeeping as a governance function that should be treated in the same manner as other governance functions such as audit and risk management. Additionally, recordkeeping and governance should be considered as symbiotic elements of a strategy. A strategy that neglects recordkeeping may not fulfil the organisation’s objectives effectively.

Attachment Style, Therapeutic Alliance and Recovery in forensic mental health: the A-STAR study and clinical research portfolio

Background: Research suggests that forensic mental health services and staff can play an important role in the recognition and intervention with attachment-related behaviours to promote engagement and recovery. There is a lack of literature exploring whether the attachment needs of forensic service-users are recognised and, associations between attachment style and factors predictive of recovery. Aims: This study aimed to examine the extent to which service-users and keyworkers agree about service-users’ attachment and to identify whether attachment was associated with service attachment, working alliance, ward climate and recovery. Methods: Twenty-two service-users from low and medium secure forensic services, completed questionnaire measures of their attachment style, service attachment, working alliance, ward climate and experiences of recovery. Nineteen keyworkers completed measures of the service-users attachment style and working alliance. Results: There was strong agreement between service-users and staff for attachment anxiety (ICC=0.71) but poor agreement for attachment avoidance (ICC=0.39). Service attachment was associated with more positive perceptions of staff support (r=0.49) and avoidant attachment was associated with lower ratings of recovery (r=-0.51). Correlations between attachment style and service attachment, working alliance and ward climate were small and non-significant. Conclusions: A focus on staff training to support recognition of the nature and impact of avoidant attachment styles is indicated. The findings suggest that interventions to enhance staff - service-user relationships may be important for service attachment and indeed promotion of a recovery focused orientation amongst service-users high in avoidant attachment may improve wellbeing and outcomes.