The use of anabolic androgenic steroids (AAS) has been associated with use of illegal or unprescribed prescription drugs, as well as different adverse psychiatric effects, such as ma-nia, psychosis and hostility. Further, there is an association between use of AAS and other different risk behaviours, including carrying guns and reckless driving. Taken together, these data suggest that there is a group of AAS users that are not elite athletes, but rather young men at risk for psychiatric illness and criminality, and who use AAS primarily for their aes-thetic effects and possibly for their psychoactive effects. The aim of this thesis is to investi-gate further the connection between use of AAS and use of other drugs, and to investigate whether the proposed side effects of AAS cause an increase in morbidity and mortality. The first study (Paper I) investigates morbidity and mortality in persons testing positive for AAS compared to persons testing negative for AAS at a doping laboratory. Paper II of this thesis studies the presence of psychoactive drugs in diseased men who tested positive for AAS upon autopsy and whether there is any difference between deceased users of AAS and deceased users of heroin or amphetamine (control group). The third article (Paper III) dis-cusses a surprising finding in paper I of increased seizures NOS in users of AAS. Paper IV and V are interview studies from an out-patient substance abuse clinic. The main findings in Paper I was that the majority of deceased users of AAS were also positive for other drugs and/or alcohol on autopsy, and that users of AAS more often than the control group had died from intentional death (suicide or homicide). The main finding of Paper II was that users of AAS were severely at risk for premature death compared to both the control group and the general population. Paper III concluded that the high prevalence of Convulsion NOS in users of AAS most likely was the result of concomitant substance abuse and withdrawal from such use. Paper IV concluded that twelve percent of the patients at the substance abuse clinic had used AAS for at least one cycle. Users of AAS had a higher risk of having been convicted of a violent offence, and users of AAS more often reported having been physically abused. In Paper V, long-terme users of AAS were found to have an increased risk for developing depression in connection with cessation of AAS use. AAS was also re-ported to be used in preparation for crime. In summary, this thesis concludes that there is a solid association between use of AAS and use of other psychotropic drugs in certain subpopulations, and that users of AAS are at risk for premature death due to unnatural causes that may be secondary to use of AAS.

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Loss of analyte from biological samples during the post-mortem interval or during storage has potentially serious implications in forensic toxicology and represents a challenge for the forensic toxicologist, especially in the interpretation of case results. The initial aim of the studies in this thesis was to evaluate the stability of some important drugs and compounds in blood under different storage conditions in order to optimize the preservation of these compounds. A second aim was to evaluate a new method of stabilizing these compounds in blood by storing them as dried blood spots on filter paper. The third aim was to investigate methods by which corrections could be made for analyte losses based on quantification of their degradation products, which would serve as markers of the former presence of the compounds even if they were no longer detectable. The background to toxicology and its classification systems is reviewed along with the most common areas of application, including forensic toxicology. Details are given of the most commonly-used matrices and of current problems facing forensic toxicologists, particularly the problem of analyte instability. The literature concerning stability of drugs and pesticides in biological samples are reviewed and discussed as well as methods applied to enhance and stabilize analytes for long storage periods. Background is provided on methodologies used in the work reported in this thesis, including extraction techniques and instrumental analysis by LC-MS/MS and GC/MS. Also, because of its importance in forensic toxicology at present validation procedures and requirements are also discussed. An initial study was made of drug stability during storage in blood samples for 1 year under conventional laboratory conditions using selected drugs from the benzodiazepine group, alprazolam, lorazepam, oxazepam and estazolam. Blank blood containing these drugs at low and high concentrations was stored in tubes at -20° C, 4°C and room temperature. Half of the tubes contained fluoride-oxalate preservative. Blood samples were analysed on the first (day zero), second and fourth days, and after one week, two weeks, one month, two months, three months, six months and one year using a method which was developed and validated for this study based on solid phase extraction (SPE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Alprazolam and estazolam were stable at -20° C and 4° C, but decreased by almost 10% at room temperature (RT) at both concentrations. Lorazepam and oxazepam were stable at 20°C but were poorly stable at 4° C and decreased by 100% at RT by the end of the 1 year period. Sodium fluoride stabilised the drugs by approximately 13% compared to unpreserved samples. The long-term stability of alprazolam and estazolam is attributed to the presence of the trizolo ring in their structures which makes the compounds more resistant to hydrolysis, the most prominent degradation reaction affecting benzodiazepines. A similar study was performed on the stability of morphine-3- and 6-glucuronide and codeine-6-glucuronide in blood and urine under the same storage conditions. These compounds were stable at -20° C, losing less than 7% but losses were higher at 4° C, up to 18% in blood and 28% in urine, and at room temperature up to 54% in blood and 78% in urine after 1 year. Sodium fluoride did not have a significant effect (<10% increase in stability). An investigation was carried out on stabilisation of hydrolytically-labile benzodiazepines and cocaine in blood during storage as dried blood spots (DBS) on filter paper. An analytical method was developed and validated for this study based on SPE and LC-MS/MS analysis. The drugs selected were flunitrazepam, temazepam, oxazepam, lorazepam, nitrazepam, diazepam and cocaine. Blood spots (100 µl blood) on Guthrie card 903 containing the drugs at 1000ng/ml were dried overnight at RT. Spots were cut out and extracted with buffer (pH 6), which was analysed with the validated method. DBS were stored in duplicate at RT, 4°C and -20°C for up to one year. Degradation of the drugs in DBS in all storage conditions was less than for the corresponding liquid blood samples stored under similar conditions. More than 80% of each analyte could be recovered from DBS after one month while 15 % cocaine and 74 % of the benzodiazepines were recovered after 1 year under all conditions. The degradation of diazepam, temazepam, chlorodiazepoxide and oxazepam by hydrolysis was studied over a 1 month period under conditions designed to accelerate the reaction (80 °C, pH 2 and 12) and the hydrolysis products 2-methylamino 5-chlorobenzophenone (MACB) and 2-amino 5-chlorobenzophenone (ACB) were analysed by a method based on SPE and LC-MS/MS which was developed and validated for this study. MACB and ACB in whole blood and urine were evaluated as indicators of the original drug concentrations. Blank blood and urine containing these compounds at 1000 ng/ml stored at high temperature (80°C) and under acidic (pH 2) and basic (pH 12) conditions at room temperature for one month. The samples were analyzed in duplicate at days 1, 2, 4, 7, 14 and 30. MACB and ACB were the main hydrolysis products and their concentrations increased as degradation of the drugs proceeded. They could be detected when the starting materials had completely disappeared. However, MACB and ACB were found to be further degraded under some of the conditions used and a further study was made of the conversion of MACB to ACB. It was concluded that the drugs studied were more sensitive to alkaline pH than to acidic pH or high temperature and that MACB and ACB can be used to confirm the original presence of these drugs in samples, especially when they have decomposed due to poor or prolonged storage conditions. A final study was made of organophosphates (OPs) and their dialkylphosphate (DAP) hydrolysis products. A new method was developed and validated for analysis of OPs and DAPs in blood samples based on SPE and GCMS after derivatization with N-tert-butyldimethylsilyl-N-methyltrifluroacetamide. The influence of sodium fluoride preservative and storage as DBS on filter paper on the stability of OPs in blood was assessed over a 3 day period at RT. With preservative, DAPs concentrations increased as degradation of the OPs proceeded and they could be detected when the parent compounds had completely disappeared. OPs in DBS showed good stability in comparison to liquid blood samples containing NAF and the parent compounds were detected at the end of the observation period. It was concluded that careful attention should be given to the storage of samples to avoid loss of analyte and erroneous interpretation of results. DBS could be an effective and inexpensive way of increasing analyte retention but routine use of preservatives without evaluation of their effects is discouraged, as these may accelerate loss of analyte.

Background: The Mental Health (Care and Treatment) (Scotland) Act 2003 reduced the role of the nearest relative, identified by a hierarchy of relationships, who previously could admit and discharge a patient as well as receive information about their care. This role is now reduced to one of receiving basic information only and the hierarchy for identification has been modernised. Service users may now nominate a named person with similar rights to service users to help protect their interests. This person cannot admit or discharge but is entitled to information and consultation about their care. If a patient has not appointed a named person, then the primary carer is appointed by default and, if there is no primary carer, the nearest relative assumes the position. Aims: To explore service users’, carers’ and professionals’ perceptions and experience of the named person provisions. Method: Twenty service users, ten carers, seven MHOs and nine professionals with influence on government policy were interviewed about their experiences. Interviews were carried out face-to-face (service users and some carers) and by telephone (carers, MHOs and policy influencers). The resulting transcripts were analysed using thematic analysis. Findings: The majority of all interviewees welcomed the introduction of the named person provisions because of the increased choice it gave service users. Service users often did not wish to nominate their nearest relative, many choosing to nominate a friend. Important factors in making a nomination were that the nominee knew the service user’s wishes and could be trusted to carry them out. Some service users chose not to nominate relatives to spare them responsibility. However, the provisions were not without their problems; uptake was perceived to be low and there were particular problems in relation to the level of understanding of the implications of a nomination by service users and of the lack of accessible information and support to increase this understanding. The imbalance of power in relationships between service users, carers and professionals was thought to impact on the autonomous choices of service users and carers. Further problems were identified with named persons appointed by default in relation to service user choice and confidentiality. Conclusion and recommendations: Although the choice is welcome to some service users, there appears to be a lack of full understanding of the role, and continued awareness-raising is required with service users, carers and professionals which should further be supported by accessible information for both service users and carers. There is currently a lack of support for carers in particular and it is recommended that this be addressed using carers’ services. It seems that many named persons are being appointed by default (itself an anomaly in Scots law) which threatens human rights, because of the lack of choice of the service user about who is involved in their care and their inability to prevent the sharing of confidential information with the default named person. The current lack of a right of service users to reject having a named person at all restricts choice and autonomy, and may further place unwanted responsibilities on carers and relatives which are difficult to remove. To ensure that service users’ rights are fully protected, the named person should become an optional nominated position and the default mechanisms removed.

In Part I, the most useful methods for the estimation of the time of death are reviewed, with special emphasis being placed on the post-mortem rate of cooling because this method is commonly used for estimating the interval after death. Theories and models of the post-mortem loss of heat from the human body are summarised and discussed. The Microwave Thermography System, a new device which is applied to this field for the first time, is described and its mode of operation is discussed. Using this device, it is possible to measure temperatures of internal organs of the body by placing the sensory elements on the skin. The reliability of the system and factors affecting the accuracy of temperature measurements made with the device are assessed and discussed. Results of a study of the cooling rate of 117 fatalities are given. All cases were studied under controlled conditions and two groups were collected in which the bodies were monitored either naked or covered with blankets. In each case, the environmental temperature as well as the temperatures at three body sites were continuously monitored over a period beginning shortly after death and ending up to 60 hours post-mortem or more. Rectal and environmental temperatures were measured with thermocouples while the temperature of the brain and liver were measured using microwave probes, therefore by non-invasive and ethically acceptable methods. The data were recorded on tape following Analogue to Digital (AD) conversion using a BBC Microcomputer. These data were processed and temperatures at the moment of death for the three body sites were estimated by extrapolation backwards. Processed data were transferred to a mainframe computer where sophisticated curve-fitting procedures were performed. These indicated that the cooling curves were adequately represented by three-term exponential equations containing six empirically derived parameters. Statistical analysis of the parameters yielded average formulae and the use of these formulae to improve the ease and accuracy of the estimation of the time of death is discussed. Lastly, suggestions for future work are given. In Part II, biochemical methods of estimating the time of death are reviewed and limitations of their use are duscussed. Steroids were selected as potential indicators of the post-morem interval by virtue of their metabolism and degradation after death. Aspects of steroid biochemistry are summarised. Various methods of steroid analysis were assessed using radioassays and thin layer chromatography. Three reversed phase chromatography systems were evaluated for separation and recovery of steroids extracted from blood, tissues and faeces. The use of different numbers of Sep-Pak C18 cartridges for the purification of steroid extracts was examined and steroid recoveries were measured and compared. The results indicated that recoveries were best when 4-6 cartridges were used. Rapid and slow procedures of enzymatic hydrolysis and acidic solvolysis of steroid conjugates were compared. A new and relatively rapid method for analysis of steroid profiles in biological samples was developed. Assessment of this method showed that steroid recoveries were improved compared to existing methods. A pilot study of the post-mortem changes in the steroid profiles of blood, tissues and faeces was carried out using the rat as a suitable and convenient animal model. Liver and adrenal tissues, faeces and blood samples collected from 30 rats either at the moment of death or 24 hours after death were analysed and their steroids were studied qualitatively and quantitatively using selctive ion monitoring GC-MS techniques. Thus chromatographic peaks were identified by comparison of their retention times and mass spectrometric characteristics with those of standards and quantitative analysis was performed. The occurrence of significant steroid changes was difficult to ascertain but some changes in the steroid profiles of the biological samples were shown to have occurred. Lastly, the practicability of this method for the estimation of time of death is discussed.

This thesis examines the use of nail as an alternative biological specimen in forensic toxicology. Nail is a difficult analytical matrix from which to extract drugs because of its tough physical composition, based on keratin. The initial part of the project investigated the use of a cryogenic grinding method for fingernail clippings. Grinding at liquid nitrogen temperatures was found to be an effective procedure and the conditions were optimised to a two or three cycle programme of freezing and grinding. Small particle sizes were obtained of approximate size 1µm. It was established that drugs could be extracted directly from nail powder with a range of solvents without the need for alkaline hydrolysis. Methanol was found to be the most effective extraction solvent, which also gave the lowest number of co-extracted interfering compounds. This procedure was subsequently used with nail samples from different types of forensic cases, including cannabis, heroin and steroid abusers. Cryogenic grinding of nail was evaluated as an extraction method for cannabinoids in nail clippings from chronic cannabis smokers. This method was also compared to the alkaline hydrolysis method. Fingernail clippings were collected with prior informed consent from volunteers attending the Edinburgh Drug Addiction Study (EDAS) clinic. The collected nail clippings were decontaminated and divided into two groups: the first group was extracted with methanol after pulverisation in a liquid nitrogen cryogenic mill; the second was extracted with ethyl acetate after hydrolysis in sodium hydroxide. In both groups deuterated cannabinoids were used as internal standards. Both sets of extracts were derivatised with BSTFA before being analysed by gas chromatography – mass spectrometry (GC-MS). Cannabidiol, ?9-tetrahydrocannabinol and 11-hydroxy-?9-tetrahydrocannibinol were quantified in both sets of extracts. 11-nor-?9 –tetrahydrocannabinol-9-carboxylic acid was only identified and quantified in the extracts resulting from the cryogenic grinding method. Cannabinoid concentrations were very low, in the range 0-4 ng/mg. These results strongly support the use of nail as a biological specimen for the detection and quantification of past exposure to cannabis, and secondly, they indicate that grinding with a cryogenic mill is a useful procedure, which yields simultaneous results for the primary psychoactive cannabinoid and its metabolites. Cryogenic grinding was then evaluated for the extraction of opioids in nail in comparison with the conventional alkaline hydrolysis method. Finger and toe nails were collected from donors with informed consent.

The aim of this study was to investigate new methods of analysis which might be applied to forensic toxicology problems including those resulting from pesticides, particularly the quaternary ammonium herbicide group, and from drugs, particularly the benzodiazepine group. In the first part of this study, an efficient method for the determination of quaternary ammonium (QA) compounds (pesticides and drugs) in human whole blood was developed. The second part of this study concerned the development of a novel sorbents for solid phase extraction using molecularly imprinted polymers (MIPs). The approach adopted was initially to synthesise a known MIP using diazepam as template then to prepare novel MIPs using other benzodiazepines and analogues of QA compounds as templates. In the first of these stages, an anti-diazepam MIP was synthesized using methacrylate acid (MAA) as the monomer and ethylene glycol dimethacrylate (EGDMA) as the cross-liner and was then ground and prepared for use as an SPE sorbent by packing it into SPE cartridges. These cartridges were used to clean up extracts of diazepam and other benzodiazepine drugs made from hair samples via a molecularly imprinted solid-phase extraction (MISPE) protocol. The MISPE method was also found to be applicable to the analysis of diazepam metabolites and other benzodiazepine drugs in addition to diazepam itself. The application of the extraction method to post-mortem hair samples yielded results that were in good agreement with ELISA data (from blood samples) and data arising from the analysis of the same blood samples using a validated in-house SPE-LC-MS-MS method. The MISPE procedure was also compared with a conventional SPE method for analysis of benzodiazepines in hair samples. The results from MISPE protocol showed better selectivity, specificity and accuracy toward diazepam (template molecule) and other benzodiazepines that display a similar resemblance to diazepam in terms of molecular structure. The MISPE procedure was found to be simpler and to offer cleaner extracts compared to a conventional SPE method.

Diss. (sammanfattning) Uppsala : University, 2005. / Härtill 4 uppsatser.

Thesis (doctoral)--Bayerische Ludwig-Maximilians-Universität, Munich, 1934.

Thesis (doctoral)--Bayerische Ludwig-Maximilians-Universität, Munich, 1934.