<p>Solid Phase Microextraction (SPME) is a popular sampling technique that can be paired with Gas Chromatography/Mass Spectrometry (GC-MS). SPME-GC-MS is used in forensic chemistry due to its simplification of the sample preparation process. Headspace-Solid Phase Microextraction (HS-SPME) is a technique where the sample is heated to generate volatiles in the headspace of the vial. A SPME fiber is then inserted into the vial and the compounds in the headspace will bind to the fiber. Total Vaporization- Solid Phase Microextraction (TV-SPME) is a technique that is derived from the HS-SPME technique. </p><p>In Chapter 1, the critical comparison of HS-SPME and TV-SPME is discussed. Samples including marijuana, essential oils, and CBD oil were utilized to compare the two techniques. The compounds of interest in marijuana are the three main cannabinoids: cannabinol (CBN), cannabidiol (CBD), and tetrahydrocannabinol (THC). The sample preparation and GC-MS parameters were kept the same for all samples to determine which SPME technique works best for these sample types and yielded the greatest sensitivity. It was found that HS-SPME shows greater sensitivity with CBN and equivalent sensitivity with essential oils, THC and CBD. </p><p>In Chapter 2, the detection of synthetic cannabinoids utilizing liquid-liquid injection as well as HS-SPME and TV-SPME is discussed. The detection of these compounds is important because this type of drug has become more prevalent in the United States because they can be chemically altered slightly so they still have the effects of a drug but can evade drug legislation. The detection of synthetic cannabinoids using liquid injection was found to be successful but detection using HS-SPME and TV-SPME was found to be unsuccessful. </p>In Chapter 3, the analyses of real and artificial saliva utilizing HS-SPME and TV-SPME is discussed. Determining the compounds present in real saliva and artificial saliva will be of importance for future research into determining if the presence of drugs in saliva can be analyzed with these techniques. The analyses of real and artificial saliva were found to be successful using HS-SPME, without derivatization, and TV-SPME, with and without derivatization. Many of the compounds present in the real saliva were detected and were confirmed to be compounds regularly found in saliva by other scientific literature.

Indiana University-Purdue University Indianapolis (IUPUI) / Solid Phase Microextraction (SPME) is a popular sampling technique that can be paired with Gas Chromatography/Mass Spectrometry (GC-MS). SPME-GC-MS is used in forensic chemistry due to its simplification of the sample preparation process. Headspace-Solid Phase Microextraction (HS-SPME) is a technique where the sample is heated to generate volatiles in the headspace of the vial. A SPME fiber is then inserted into the vial and the compounds in the headspace will bind to the fiber. Total Vaporization- Solid Phase Microextraction (TV-SPME) is a technique that is derived from the HS-SPME technique. In Chapter 1, the critical comparison of HS-SPME and TV-SPME is discussed. Samples including marijuana, essential oils, and CBD oil were utilized to compare the two techniques. The compounds of interest in marijuana are the three main cannabinoids: cannabinol (CBN), cannabidiol (CBD), and tetrahydrocannabinol (THC). The sample preparation and GC-MS parameters were kept the same for all samples to determine which SPME technique works best for these sample types and yielded the greatest sensitivity. It was found that HS-SPME shows greater sensitivity with CBN and equivalent sensitivity with essential oils, THC and CBD. In Chapter 2, the detection of synthetic cannabinoids utilizing liquid-liquid injection as well as HS-SPME and TV-SPME is discussed. The detection of these compounds is important because this type of drug has become more prevalent in the United States because they can be chemically altered slightly so they still have the effects of a drug but can evade drug legislation. The detection of synthetic cannabinoids using liquid injection was found to be successful but detection using HS-SPME and TV-SPME was found to be unsuccessful. In Chapter 3, the analyses of real and artificial saliva utilizing HS-SPME and TV-SPME is discussed. Determining the compounds present in real saliva and artificial saliva will be of importance for future research into determining if the presence of drugs in saliva can be analyzed with these techniques. The analyses of real and artificial saliva were found to be successful using HS-SPME, without derivatization, and TV-SPME, with and without derivatization. Many of the compounds present in the real saliva were detected and were confirmed to be compounds regularly found in saliva by other scientific literature.

Hemp belongs among important plants with a long industrial tradition. However, its first use was originally for healing. In medicine, it is mainly applied thanks to the content of many biologically active substances, such as cannabinoids. This thesis is concerned with the use of hemp in acne preparations. The theory focuses mainly on the characterization of hemp and its active substances. The other chapters deals with cosmetic preparations and acne itself, and Propionibacterium acnes as well, which is involved. Within the experimental part, the extracts of two hemp varieties (Finola and Bialobrzeskie) were characterized in terms of antioxidant and cannabinoid presence. The cosmetic preparations with hemp extract content were prepared including a complete cleansing set for acne skin, concurrently commercial products of this sort were purchased. In cosmetic preparations and the extracts their inhibitory effect against Propionibacterium acnes was studied as well. Also Micrococcus luteus, Escherichia coli and Candida glabrata were tested. The results show that dried hemp flower extracts contain the greatest amount of active substances, so they have a higher antioxidant and antimicrobial effect. The presence of individual cannabinoids differed mainly depending on the variety. All hemp extracts had effect against gram-positive bacteria, in cosmetic preparations it was lower. Nevertheless hemp antibacterial effect has been proven.

The thesis occupies with the use of hemp in cosmetics and in food processing. In the theoretical part hemp was characterized by the content of active substances. In the experimental part the thesis is concerned with characterization of two different hemp varieties Carmagnola and Ferimon in terms of presence of active substances. The next part focuses on preparation of cosmetic with hemp content. In cosmetic preparations and in the extracts the inhibitory effect against Propionibacterium acnes, Micrococcus luteus, Escherichia coli and Candida glabrata was tested. The content of fatty acids and vitamin E was determined in hemp seed. The results show that dried hemp flower extract contains the largest amount of active substances and has the greatest inhibitory effect against all tested bacteria except Candida there was no effect. Cosmetic preparations did not show almost any inhibitory effect because of low concentration of active substances. The hemp seed contained high amount of PUFA and essential fatty acids. Vitamin E was found out in all of three hemp oils.

A cannabinoid refers to any natural or synthetic compound that interacts with the CB1 and CB2 receptors. There are currently three different groups of cannabinoids: endogenous cannabinoids, phytocannabinoids and synthetic cannabinoids. The most common phytocannabinoid is delta-9-tetrahydrocannabinol (THC), which is the active component in the Cannabis sativa or marihuana plant1–3. Two examples of synthetic cannabinoids that are present in case reports from 2012 to 2018 are AB-FUBINACA and AB-PINACA4–7. THC and synthetic cannabinoids are commonly encountered drugs in forensic toxicology cases, therefore, being able to extract these compounds and their metabolites is imperative for toxicological interpretation. There are a variety of commercially available sample preparation techniques for these analytes. Companies such as UCT, Biotage, Millipore-Sigma, Tecan, and Thermo Fisher Scientific manufacture these products. The focus of this research was to evaluate these techniques for their cleanliness, efficiency and cost effectiveness. Sample preparation techniques are designed to remove the different components of the matrix and other prescription or illicit substances present in the sample that could interfere with the assay, increase the analyte recovery, extraction efficiency, decrease variability, and clean-up the sample to allow for less instrument downtime and longer column life8. This study focused on comparing a liquid-liquid extraction (LLE), solid phase extraction (SPE), and supported liquid extraction (SLE). The primary purpose of this study was to develop and validate the three above mentioned sample preparation techniques for the analysis of THC, 11-hydroxy-THC, 11-nor-9-carboxy-THC (THCCOOH), AB-FUBINACA, AB-FUBINACA metabolite 3, and AB-PINACA in blood and urine. Parameters assessed followed Academy Standards Board (ASB) Standard 036, Standard Practices for Method Validation in Forensic Toxicology, including recovery, suppression, and matrix effects. For urine and blood analysis, the calibration range was determined to be 1 ng/mL to 50 ng/mL for all three techniques. Urine recovery was highest for the LLE method, with all compounds having a recovery greater than 50%. The SLE method had the lowest LOQ results for urine, with 0.5 ng/mL for 11-hydroxy-THC and THCCOOH, 0.75 ng/mL for THC, AB-FUBINCA and AB-FUBINACA metabolite 3, and 1 ng/mL for AB-PINACA. Ion suppression was reduced using the SLE method for urine along with having the shortest sample preparation time of 1 hr for up to 48 samples. For blood analysis, the LLE method had the greatest recovery of all analytes. The LLE method also had reduced suppression and matrix effects compared to the SPE method. Sample preparation was shorter for the SPE method, consuming 2 hrs for an average sample batch, compared to 4 hrs for the LLE method, which included a 2 hr freezing step. In conclusion, for urine analysis, all three sample preparation techniques were acceptable for the analysis of THC, synthetic cannabinoids, and their metabolites, with the SLE method being the preferred method. For blood analysis a LLE and SPE method were developed and are adequate for the analysis of THC, synthetic cannabinoids, and their metabolites, with the LLE method being the preferred method.

Despite efforts to control synthetic cannabinoids, clandestine manufacturers continue to modify their structures to avoid legal consequences, creating an ever-changing analytical target for forensic laboratories (1). Forensic toxicology laboratories often lack the needed resources or do not have the capabilities to test for these compounds and metabolites, requiring specimens to be submitted to reference laboratories (2). Drug stability can be affected by long storage times, temperature and preservatives (3). Although these factors can be controlled, systematic research is necessary to identify their impacts on the stability of these new synthetic cannabinoids that are continually emerging. The purpose of this research is to assess the stability of 17 synthetic cannabinoids in human whole blood and 10 synthetic cannabinoid metabolites in human urine using liquid chromatography-tandem mass spectrometry (LC-MS/MS) over thirty-five weeks. The analysis methods were validated in accordance to the Academy Standards Board (ASB) method validation guidelines for quantitative analysis and stability evaluation of the following analytes in blood: 4-cyano CUMYL-BUTINACA, ADB-PINACA, EMB-FUBINACA, JWH-250, MO-CHMINACA, 5-fluoro-3,5-ABPFUPPYCA, 5-fluoro ADB-PINACA, APP-PICA, CUMYL-THPINACA, PB-22, XLR11, 5-fluoro PY-PINACA, MDMB-FUBICA, MEP-CHMICA, NM2201, RCS-8, and UR144. The stability analysis in urine includes the following metabolites: 5-fluoro MDMB-PICA metabolite 7, 5-fluoro PB-22 3-carboxyindole, AB-FUBINACA metabolite 3, ADB-PINACA N-(4-hydroxypentyl), ADB-PINACA pentanoic acid, UR-144 Degradant N-pentanoic acid, PB-22 N-(5-hydroxypentyl), MDMB-FUBICA metabolite 3, UR-144 N-(5-hydroxypentyl), and JWH-250 N-pentanoic acid. Research samples were prepared by spiking with certified reference standards (Cayman Chemical, Ann Arbor, MI, USA) of each select synthetic cannabinoid in certified drug-free human whole blood (Boston Medical Center, Boston, MA, USA; Biological Specialty Corporation, Colmar, PA) and drug-free urine that was received as donations following the approved Institutional Review Board guidelines (Boston University School of Medicine, Boston, MA, USA). Blood samples were aliquoted into 6 mL BD Vacutainer Plastic Collection Tubes (Fisher Scientific, Waltham, MA, USA) and urine samples were stored in 15 mL Falcon Conical Centrifuge Tubes (Fisher Scientific, Waltham, MA, USA). Stability under room temperature (20ºC), refrigerator (4ºC), and freezer (-20ºC) at low and high concentrations were evaluated at select time points. A 5% solution of potassium oxalate and sodium fluoride or ethylenediaminetetraacetic acid (EDTA) was added to the preserved blood samples by the manufacturer prior to storage. The anticoagulant, potassium oxalate, was only added in solution to the preserved samples whereas none was added to the nonpreserved samples. Short-term urine samples were preserved with 1% of sodium fluoride prior to storage. Extraction of analytes was conducted using supported-liquid extraction (SLE) ISOLUTE 1 mL cartridges (Biotage, Charlotte, NC, USA) and reconstituted in 100 μL of 50:50 mixture of 0.1% formic acid in millipore deionized water and 0.1% formic acid in acetonitrile (Fisher Scientific, Waltham, MA, USA). Analysis was performed in triplicate using a reverse-phase C18 column (Waters XBridge C18 3.5 μM, 2.1 x 50 mm, Milford, MA, USA) on the Shimadzu Prominence Ultra-Fast Liquid Chromatography (UFLC, Kyoto, Japan) with SCIEX 4000 Q-Trap Electrospray Ionization Tandem Mass Spectrometry (ESI/MS/MS, Waltham, MA, USA) in positive ionization mode. The total run time was 8 minutes with a flow rate of 0.6 mL/min and injection volume of 10 μL. Linear calibration curves for each analyte with the exception of a quadratic regression for PB-22, all had acceptable R2 values > 0.99 using a weighting factor of 1/x. A linear dynamic range of 0.5 – 25 ng/mL was used for all analytes in blood except for NM2201 and APP-PICA with a limit of quantitation (LOQ) of 0.1 ng/mL and MO-CHMINACA with a working range of 0.5 – 15 ng/mL. A linear working range of 5 – 40 ng/mL was utilized for all metabolites in urine. No signs of carryover were observed. In general, analytes were considered stable if the average area ratio between the analyte and internal standard at the time point was within ± 20% of the average area ratio response at time point zero. In some cases, it was necessary to evaluate the complete picture of the stability data by reviewing analyte area, concentration, and overall stability data trend between timepoints at the low and high concentrations. In certain situations, an analyte was considered stable even if specific timepoints for a concentration were outside the ±20% range. For example, in cases where one concentration at a timepoint was within the ±20% range and the other concentration fell within ±30% range the analyte was considered stable overall. Long-term stability results revealed that all synthetic cannabinoids were stable at 21 to 35 weeks in frozen blood preserved with sodium fluoride except for APP-PICA. The preservatives are recommended to be added to blood to reduce the possibility of matrix inferences and minimize detrimental impacts on the stability of synthetic cannabinoids. Analytes experienced lower degradation in the order of samples that were kept frozen, refrigerated, and then at room temperature. Blood analytes that were stable up to 35 weeks in freezer generally had a core structure of a carbonyl substituent on a pyrazole or pyrrole with surrounding nonpolar groups; whereas compounds with two polar carbonyl functional groups present were found to experience degradation much earlier at 1 week or less in room temperature and refrigerator storage conditions. 5-fluoropentyl analogs, like XLR11 and 5-fluoro ADB-PINACA, in comparison to their counterpart analyte, UR144 and ADB-PINACA, were unstable at earlier time points under all storage conditions. Instability in the majority of the urine metabolites was not observed until after 9 weeks and was generally consistent across all storage conditions. The validated methods demonstrate a sensitive and reliable way to positively identify 17 different synthetic cannabinoids in human whole blood and 10 synthetic cannabinoid metabolites in urine for rapid time stability analysis at various storage conditions. The use of SLE improved sample preparation efficiency by decreasing the extraction time from 1 hour to 30 minutes compared to traditional extraction methods, such as solid-phase extraction (SPE) and liquid-liquid extraction (LLE). Further studies into additional matrices, such as oral fluid, longer storage times, and other emerging synthetic cannabinoid analytes would expand the scope of this research.

No description available.

Decades of research has provided evidence for the role of the endocannabinoid system in human health and disease. This versatile system, consisting of two receptors (CB1 and CB2), their endogenous ligands (endocannabinoids), and metabolic enzymes has been implicated in a wide variety of disease states, ranging from neurological disorders to cancer. CB2 has gained much interest for its beneficial immunomodulatory role that can be obtained without eliciting psychotropic effects through CB1. Recent studies have shed light on a protective role of CB2 in cardiovascular disease, an ailment which currently takes more lives each year in Western countries than any other disease or injury. By use of CB2 knockout mice and CB2-selective ligands, knowledge of how CB2 signaling affects atherosclerosis and ischemia has been acquired, providing a major stepping stone between basic science and translational clinical research. Here, we summarize the current understanding of the endocannabinoid system in human pathologies and provide a review of the results from preclinical studies examining its function in cardiovascular disease, with a particular emphasis on possible CB2-targeted therapeutic interventions to alleviate atherosclerosis.

Many South African psychiatrists, and particularly those working in psychiatric hospitals with Black and Coloured patients, have the firm clinical impression that in many of these patients acute psychotic illness is associated with the abuse of cannabis. Most of the previous work in this field had been done by clinicians of Eastern countries where the use of cannabis has been endemic for thousands of years. However, those workers were handicapped because they lacked both the sophisticated techniques for standardized psychiatric evaluation and the availability of an assay to confirm cannabis use. It was decided to investigate acute psychoses in Cape Coloured males admitted to Valkenberg Hospital with the following aims: i. To identify a cohort of acutely psychotic patients who had recently been using cannabis and to compare them with a matched control group who were free of any drugs. The recently available EMITR immunochemical analytical technique was used for the detection of urinary cannabinoids. To exclude the contribution of other psychotropic agents to the aetiology of the psychoses, gas chromatography was performed to detect ethanol and thin-layer chromatography to screen for other psychotropic agents. ii. To assess the comprehensive mental state of patients on admission and then again after a 7-10 day period the Present State Examination (PSE), a well validated and standardized diagnostic instrument, was used. iii. To determine serum creatinine phosphokinase (CPK) and serum lactate dehydrogenase (LDH) levels (indicators of muscle damage) in view of the published reports of elevated levels in psychotic patients.

Synthetic cannabinoids (SCs), present a multitude of problems in terms of maintaining up-to-date, reliable, specific, and sensitive methods of detection. Synthetic cannabinoids are novel psychoactive substances originally synthesized for medical use and research purposes. Abuse of these compounds, however, has demonstrated a variety of effects ranging from euphoria to aggressive behavior and loss of consciousness. The most dangerous reported result of synthetic cannabinoids use has been death. The number of synthetic cannabinoid compounds detected drastically increased from two to over 80 compounds within six years. The marketing of these compounds, similar naming, and described pharmacological interactions, create the dangerous and very false perception that SCs are similar to, or the same as, tetrahydrocannabinol in cannabis products. This research focused on the development of a method to detect and quantify seven synthetic cannabinoids in urine, plasma, and gummy bears. The seven synthetic cannabinoids studied include XLR-11, AB-PINACA 5-pentanoic acid metabolite, UR-144 5-pentanoic acid metabolite, 5F-PB-22, AM-2201 4-hydroxypentyl metabolite, JWH-018, and JWH-018 5-hydroxypentyl metabolite. Sample preparation methods and a two dimensional liquid chromatography tandem mass spectrometry method were optimized and developed for analysis of the seven SCs in each matrix. The method was successfully applied to 17 authentic urine case samples previously screened positive for synthetic cannabinoids and a calibration curve for each matrix was generated from spiked samples at varying concentrations. Utilizing two-dimensional (2D) chromatography for the analysis of synthetic cannabinoids allowed for a novel approach to be employed. With this method, 100% organic samples were analyzed with improved resolution and increased sensitivity. The sample preparation method for the urine and plasma samples included a protein precipitation technique with acid followed by solid phase extraction (SPE) on a mixed-mode reversed phase strong anion exchange sorbent. The spiked gummy bear samples were prepared in 50% methanol in water, dissolved by heating, and extracted with SPE on the same sorbent used for the urine and plasma samples. A 200µL injection of the 100% MeOH extracts was injected into 2D-LC-MS/MS for analysis with a loading and diluting solvent consisting of water and 2% ammonium hydroxide and elution solvents containing water or methanol with 0.5% formic acid. These conditions were optimized with an automated method development protocol assessing various conditions such as mobile phase solvents, pH additives, and trap column chemistries. The final chromatography method utilized an ACQUITY ultra performance liquid chromatography (UPLC) ethylene bridged hybrid (BEH) C8 2.1 x 30mm, 10µm trap column and an ACQUITY UPLC high strength silica with tri-functional C18 bonding (HSS T3) analytical column 2.1 x 150mm, 1.7µm. The urine calibration curve produced had a linear dynamic range (LDR) of 0.05-2.5ng/mL for UR-144 5-COOH and AB-PINACA 5-COOH and 0.05-5ng/mL for the other five synthetic cannabinoids. R2 values included 0.992 and 0.993 for UR-144 5-COOH and AB-PINACA 5-COOH, respectively and 0.995 or above for the other five compounds. Synthetic cannabinoids were detected at varying concentrations in all 17 urine case samples. Analysis of plasma and gummy bear samples was also successfully carried out. Plasma calibration curves had a LDR 0.05-10ng/mL with all R2 values above 0.995. Gummy bear calibration curves produced a LDR of 0.05-10ng/mL or 0.05-2.5ng/mL with R2 values over 0.995. All extraction recovery values were greater than 80% with the exception of 63% recovery for AB-PINACA 5-COOH in the gummy bear matrix. Suppression effects of 8%, 19%, and 6.6% were observed for urine, plasma, and gummy bears, respectively. Relatively low recovery values, reduced linear dynamic ranges, and suppression matrix effects for the carboxylic acid analytes assessed in this research suggested an alternative approach may be more successful for the analysis of these particular compound types in all three matrices. Overall, a sensitive, specific, and reliable method was developed with low limits of detection and quantification for efficient and rapid analysis of compounds at trace levels utilizing 2D-LC-MS/MS.