Chromatographic analysis and survey studies to evaluate the emerging drugs of synthetic cannabinoids in Scotland and Saudi Arabia

Synthetic cannabinoid receptor agonists or more commonly known as synthetic cannabinoids (SCs) were originally created to obtain the medicinal value of THC but they are an emerging social problem. SCs are mostly produced coated on herbal materials or in powder form and marketed under a variety of brand names, e.g. “Spice”, “K2”. Despite many SCs becoming controlled under drug legislation, many of them remain legal in some countries around the world. In Scotland, SCs are controlled under the Misuse of Drugs Act 1971 and Psychoactive Substances Act 2016 that only cover a few early SCs. In Saudi Arabia, even fewer are controlled. The picture of the SCs-problem in Scotland is vague due to insufficient prevalence data, particularly that using biological samples. Whilst there is evidence of increasing use of SCs throughout the world, in Saudi Arabia, there is currently no data regarding the use of products containing SCs among Saudi people. Several studies indicate that SCs may cause serious toxicity and impairment to health therefore it is important to understand the scale of use within society. A simple and sensitive method was developed for the simultaneous analysis of 10 parent SCs (JWH-018, JWH-073, JWH-250, JWH-200, AM-1248, UR-144, A-796260, AB-FUBINACA, 5F-AKB-48 and 5F-PB-22) in whole blood and 8 corresponding metabolites (JWH-018 4-OH pentyl, JWH-073 3-OH butyl, JWH-250 4-OH pentyl, AM-2201 4-OH pentyl, JWH-122 5-OH pentyl, JWH-210 5-OH pentyl, 5F-AKB-48 (N-4 OH pentyl), 5F-PB-22 3-carboxyindole)in urine using LLE and LC-MS/MS. The method was validated according to the standard practices for method validation in forensic toxicology (SWGTOX, May 2013). All analytes gave acceptable precision, linearity and recovery for analysing blood and urine samples. The method was applied to 1,496 biological samples, a mixture of whole blood and urine. Blood and/or urine samples were analysed from 114 patients presenting at Accident and Emergency in Glasgow Royal Infirmary, in spring 2014 and JuneDecember 2015. 5F-AKB-48, 5F-PB-22 and MDMB-CHMICA were detected in 9, 7 and 9 cases respectively. 904 urine samples from individuals admitted to/liberated from Scottish prisons over November 2013 were tested for the presence of SCs. 5F-AKB-48 (N-4 OH pentyl) was detected in 10 cases and 5F-PB-22 3-carboxyindole in 3 cases. Blood and urine samples from two post-mortem cases in Scotland with suspected ingestion of SCs were analysed. Both cases were confirmed positive for 5F-AKB-48. A total of 463 urine samples were collected from personnel who presented to the Security Forces Hospital in Ryiadh for workplace drug testing as a requirement for their job during July 2014. The results of the analysis found 2 samples to be positive for 5F-PB-22 3carboxyindole. A further study in Saudi Arabia using a questionnaire was carried out among 3 subpopulations: medical professionals, members of the public in and around smoking cafes and known drug users. With regards to general awareness of Spice products, 16%, 11% and 22% of those participants of medical professionals, members of the public in and around smoking cafes and known drug users, respectively, were aware of the existence of SCs or Spice products. The respondents had an overall average of 4.5% who had a friend who used these Spice products. It is clear from the results obtained in both blood and urine testing and surveys that SCs are being used in both Scotland and Saudi Arabia. The extent of their use is not clear and the data presented here is an initial look into their prevalence. Blood and urine findings suggest changing trends in SC use, moving away from JWH and AM SCs to the newer 5F-AKB-48, 5-F-PB-22 and MDMBCHMICA compounds worldwide. In both countries 5F-PB-22 was detected. These findings clarify how the SCs phenomenon is a worldwide problem and how the information of every country regarding what SCs are seized can help and is not specific for that country. The analytes included in the method were selected due to their apparent availability in both countries, however it is possible that some newer analytes have been used and these would not have been detected. For this reason it is important that methods for testing SCs are updated regularly and evolve with the ever-changing availability of these drugs worldwide. In addition, there is little published literature regarding the concentrations of these drugs found in blood and urine samples and this work goes some way towards understanding these.

Conservation value, biodiversity value and methods of assessment in regenerating and human disturbed tropical forest

Although the value of primary forests for biodiversity conservation is well known, the potential biodiversity and conservation value of regenerating forests remains controversial. Many factors likely contribute to this, including: 1. the variable ages of regenerating forests being studied (often dominated by relatively young regenerating forests); 2. the potential for confounding on-going human disturbance (such as logging and hunting); 3. the relatively low number of multi-taxa studies; 4. the lack of studies that directly compare different historic disturbances within the same location; 5. contrasting patterns from different survey methodologies and the paucity of knowledge on the impacts across different vertical levels of rainforest biodiversity (often due to a lack of suitable methodologies available to assess them). We also know relatively little as to how biodiversity is affected by major current impacts, such as unmarked rainforest roads, which contribute to this degradation of habitat and fragmentation. This thesis explores the potential biodiversity value of regenerating rainforests under the best of scenarios and seeks to understand more about the impact of current human disturbance to biodiversity; data comes from case studies from the Manu and Sumaco Biosphere Reserves in the Western Amazon. Specifically, I compare overall biodiversity and conservation value of a best case regenerating rainforest site with a selection of well-studied primary forest sites and with predicted species lists for the region; including a focus on species of key conservation concern. I then investigate the biodiversity of the same study site in reference to different types of historic anthropogenic disturbance. Following this I investigate the impacts to biodiversity from an unmarked rainforest road. In order to understand more about the differential effects of habitat disturbance on arboreal diversity I directly assess how patterns of butterfly biodiversity vary between three vertical strata. Although assessments within the canopy have been made for birds, invertebrates and bats, very few studies have successfully targeted arboreal mammals. I therefore investigate the potential of camera traps for inventorying arboreal mammal species in comparison with traditional methodologies. Finally, in order to investigate the possibility that different survey methodologies might identify different biodiversity patterns in habitat disturbance assessments, I investigate whether two different but commonly used survey methodologies used to assess amphibians, indicate the same or different responses of amphibian biodiversity to historic habitat change by people. The regenerating rainforest study site contained high levels of species richness; both in terms of alpha diversity found in nearby primary forest areas (87% ±3.5) and in terms of predicted primary forest diversity from the region (83% ±6.7). This included 89% (39 out of 44) of the species of high conservation concern predicted for the Manu region. Faunal species richness in once completely cleared regenerating forest was on average 13% (±9.8) lower than historically selectively logged forest. The presence of the small unmarked road significantly altered levels of faunal biodiversity for three taxa, up to and potentially beyond 350m into the forest interior. Most notably, the impact on biodiversity extended to at least 32% of the whole reserve area. The assessment of butterflies across strata showed that different vertical zones within the same rainforest responded differently in areas with different historic human disturbance. A comparison between forest regenerating after selective logging and forest regenerating after complete clearance, showed that there was a 17% greater reduction in canopy species richness in the historically cleared forest compared with the terrestrial community. Comparing arboreal camera traps with traditional ground-based techniques suggests that camera traps are an effective tool for inventorying secretive arboreal rainforest mammal communities and detect a higher number of cryptic species. Finally, the two survey methodologies used to assess amphibian communities identified contrasting biodiversity patterns in a human modified rainforest; one indicated biodiversity differences between forests with different human disturbance histories, whereas the other suggested no differences between forest disturbance types. Overall, in this thesis I find that the conservation and biodiversity value of regenerating and human disturbed tropical forest can potentially contribute to rainforest biodiversity conservation, particularly in the best of circumstances. I also highlight the importance of utilising appropriate study methodologies that to investigate these three-dimensional habitats, and contribute to the development of methodologies to do so. However, care should be taken when using different survey methodologies, which can provide contrasting biodiversity patterns in response to human disturbance.