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.

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.

Synthetic cannabinoids are recreational drugs designed to mimic the effects of Δ9-tetrahydrocannabinol (THC), the main psychoactive component present in cannabis. These drugs exhibit severe toxic effects upon consumption due to their high binding affinity and potency at the cannabinoid receptors (CB1 and CB2). Synthetic cannabinoids have proliferated over the last decade and become a major public health and analytical challenge, critically impacting the clinical and forensic communities. Indazole carboxamide and indole carboxamide class synthetic cannabinoids have been particularly rampant, and are the compound classes most frequently reported to governmental agencies worldwide. However, the metabolic and pharmacological properties of many of these compounds remains unknown. Elucidating these characteristics allows members of the clinical and forensic communities to identify causative agents in patient samples, as well as render conclusions regarding their toxic effects. The aim of this research study was to assess the in vitro Phase I metabolic profile of five synthetic cannabinoids and report the major metabolites identified; compounds evaluated included MDMB-CHNINACA; APP-CHMINACA (PX-3); 5F-APP-PICA (PX-1); 5F-MDMB-PINACA (5F-ADB); and FUB-AMB. These analytes were incubated for 120 minutes with human liver microsomes, followed by analysis of the extracts via ultra high performance liquid chromatography – tandem mass spectrometry (UHPLC-MS/MS). The high-resolution mass spectrometry tool utilized (quadrupole-time of flight mass spectrometry, QTOF) allowed for a thorough characterization of the metabolites, including the assignment of a chemical formula and structure, and accurate mass. The metabolic stability and kinetic profiles of 5F-ADB and FUB-AMB were evaluated by aliquoting the incubation samples at various time points throughout the procedure. It was observed that these compounds were metabolized rapidly, resulting in short half-lives and relatively elevated metabolic clearances. A variety of metabolites were identified for most of the species studied, and this was dependent on the chemical structure of the parent molecule. The major metabolites identified overall for the species were products of amide or ester hydrolysis; hydroxylation (including polyhydroxylation) of the pentyl side chain or cyclohexylmethyl moiety; and oxidative defluorination. It is proposed that these metabolites (especially analyte-specific metabolite) be included in laboratory assay panels to facilitate unequivocal identification of the synthetic cannabinoid agent of interest. For select compounds (5F-ADB and FUB-AMB), authentic forensic human blood samples which screened positive for these analytes were provided by a renowned forensic toxicology laboratory. These samples were tested to verify that the major metabolites identified in the in vitro studies were also present in blood in vivo; the resultant data from the 5F-ADB and FUB-AMB samples showed that the major hydroxylated and hydrolysis metabolite, respectively, were present in greater abundance than the parent molecule, which was most often absent or not present in an appreciable quantity. Additionally, it was observed in the time studies of 5F-ADB and FUB-AMB that the metabolites containing carboxylic acid functional groups were detected in incubation samples longer than the hydroxylated metabolites, potentially indicative of longer detection windows in human samples. These findings have important toxicological implications; many synthetic cannabinoid metabolites, including those identified in this study may have pharmacological activity and contribute to a drug user’s overall impairment profile; identifying them in blood in the absence of parent compound can point to the causative agent. The results demonstrate that it is imperative that synthetic cannabinoid assays screen for known pharmacologically active metabolites; this is particularly important for drugs with short half-lives. The results of this research can be applied to the prediction of metabolic pathways for synthetic cannabinoids as well as non-drug substances with similar structural elements whose metabolic profile has not yet been elucidated, and whose pharmacological activity is currently unknown. Additionally, the results provide reference standard manufacturers and research scientists with further insight into the metabolic products of synthetic cannabinoids and related compounds for the synthesis of materials for the development of laboratory assays. / Chemistry

Syftet med denna studie är att utröna varför personer väljer att använda syntetiska cannabinoider. Vidare ämnade studien undersöka om personer som använder syntetiska cannabinoider också använder andra droger. Samtidigt har studien målet att ta reda på i vilken utsträckning drogens laglighet inverkar vid valet att använda drogen. Studien är kvantitativ. Undersökningen utfördes genom en webbenkät som besvarades av totalt 356 respondenter. Av dessa hade 171 personer använt syntetiska cannabinoider. I resultatet framkom att anledningarna till att använda drogen är flera, men de främsta visade sig vara laglighet, nyfikenhet, tillgänglighet, ruset samt att det ej visar sig vid urinprov. Personer som använder syntetiska cannabinoider har en tendens att också använda flera andra droger. Drogens laglighet visade sig ha en relativt stor inverkan på valet att använda den. Det visade sig också att män i större utsträckning använder syntetiska cannabinoider i jämförelse med kvinnor. / The purpose of this study is to examine why people choose to use synthetic cannabinoids. Furthermore, the study intended to investigate whether people who use synthetic cannabinoids also using other drugs. Meanwhile the study has the objective to find out to what extent the drug affects the legality of the choice to use the drug. The study is quantitative. The survey was conducted through an online survey and was answered by a total of 356 respondents. Of these, 171 people used synthetic cannabinoids. The result showed that the reason for using the drug are several, but the main proved were legality, curiosity, availability, intoxication and that it is not revealed by urine testing. People using synthetic cannabinoids have a tendency to also use several other drugs. The legacy of the drug was found to have a relatively large impact on the decision to use it. It was also found that men in greater occurrence use synthetic cannabinoids compared to women.

Introduction: Over recent decades, there has been an increase in the availability and use of Novel Psychoactive Substances (NPS) all over the world. They include several classes of chemicals that mimic the effects of illicit drugs and have been purposefully introduced into the market to circumvent or undermine the purpose of legal regulation. Currently, there is information lacking on the pharmacology of these substances; however, the increasing number of cases and outbreaks of intoxications/deaths is becoming a cause for deepening concern. Multi-disciplinary research in the fields of biology, chemistry, clinical medicine and web analysis is needed to develop responses against this tidal wave. Aim: The overall aim of this project is to gain insights into pharmacological, neurochemical and molecular properties of selected NPS to provide a reliable background needed for detection, assessment, and management of NPS-related harms. A range of approaches and methodologies was employed and a spectrum of different fields of knowledge has been engaged to gain some understanding into the complex multi-faceted phenomenon of NPS. Methods: Different substances have been selected as targets for the present project according to the clinical pattern of toxicity raised by their worldwide use and the lack of scientific knowledge available about them. The methods employed were: in vitro quantitative autoradiography (to evaluate the binding properties of the novel SCs BB-22, 5F-PB-22, 5F-AKB-48 and STS-135 at the cannabinoid receptor type 1 and N-methyl-D-aspartate receptor; and the binding properties of the synthetic stimulants 5-IT and 2-DPMP at the dopamine transporter in rat brain slices); in vitro Fast Scan Cyclic Voltammetry (to assess the effects of BB-22 on evoked dopamine efflux and dopamine re-uptake half-life in nucleus accumbens brain slices); in vivo microdialysis (to monitor dopamine release in terminal areas of the reward system after acute administration of the synthetic cannabinoids BB-22, 5F-PB-22, 5F-AKB-48 and STS-135; the dieting aid compound 2,4-DNP; the synthetic stimulants 2-DPMP and D2PM in freely moving animals); in silico molecular docking (to investigate the intermolecular interactions of the SCs BB-22, 5F-PB-22, 5F-AKB-48 and STS-135, and other referent compounds, with a homology model of the rodent cannabinoid receptor type 1 (CB1R) and the crystal structure of the human CB1R); and a web-based analysis approach (to analyse the information provided by a range of fora communities on 4,4'-DMAR use, additionally critical reviewing the available evidence-based literature on this topic). Results: Our in vitro quantitative autoradiography studies, confirmed that the index compounds BB-22, 5F-PB-22, 5F-AKB-48 and STS-135, behave as highly potent CB1R ligands able to compete with the radioligand [3H]CP-55,940 in cortical and striatal brain slices. On the other hand, all synthetic cannabinoids tested were unable to compete with the radioligand [3H]MK-801 in the same cerebral areas, rejecting the hypothesis of their potential binding to the N-methyl-D-aspartate receptor (NMDAR) at all concentrations investigated. Consistent with previous in vitro studies, 5-IT and 2-DPMP behaved as highly potent dopamine transporter (DAT) ligands able to compete with the radioligand [125 I]RTI-121 in a concentration-dependent way in the Caudate Putamen (CPu) and Nucleus Accumbens (NAc) brain slices. Notably, 2-DPMP was able to displace the radioligand in both cerebral regions, starting from lower concentrations compared to 5-IT. In vitro Fast Scan Cyclic Voltammetry findings demonstrated that local application of the synthetic cannabinoid BB-22 in brain slices, was unable to change evoked dopamine efflux and dopamine reuptake time-constant in the NAc shell at any doses tested. The results obtained would suggest the relative contributions of complex neuronal circuits, either within or outside the NAc, whose modulation would interfere with the interactions between BB-22 and dopaminergic neurons and represent critical pathways accounting for some of the rewarding properties of BB-22 exposure. In vivo microdialysis outcomes suggested that all SCs tested could increase dopamine release in the NAc shell at specific doses, while no changes in dopamine output were observed in other areas of the reward system, namely NAc core and medial prefrontal cortex (mPFCx) after BB-22 administration. These outcomes provided a circumstantial pre-clinical evidence for a greater putative abuse liability of SCs compared to the natural compound found in cannabis (Δ9‐THC). Furthermore, the acute treatment with 2,4-DNP did not cause any change in dopamine release in the NAc shell and CPu rejecting the hypothesis of psychoactivity of this substance at the dose tested. On the other hand, the synthetic stimulant 2-DPMP elicited a comparable increase of dopamine (DA) release in the NAc shell and CPu at the higher doses tested, while D2PM caused a selective increase of DA release in the NAc shell, providing a circumstantial preclinical evidence for a putative abuse liability of this compound at the highest dose assessed. The in silico molecular docking studies demonstrated that the SCs BB-22, 5F-PB-22, 5F-AKB-48 and STS-135 interact with CB1 receptor residues that, according to previous mutation and computational studies, are considered crucial for synthetic cannabinoid binding recognition. Additionally, they share some interacting residues with other aminoalkylindole derivatives (e.g. WIN-55,212-2). The web-based analysis focused on 4,4'-DMAR, suggested that fora members co-operate in exchanging an extensive body of knowledge about this drug, and the recurring topics of discussion include: routes of administration and dosages; desired and undesired effects; comparison and association with other drugs and medications; overall impression; provision of harm reduction advice. This approach has been useful to better understand some of the clinical and psychopharmacological issues pertaining to 4,4'-DMAR. Conclusions: Overall, these studies provided new pharmacological, neurochemical and molecular knowledge on a range of Novel Psychoactive Substances essential for identifying potential therapeutical approaches against their use/abuse. The novelty of this project lies in the adoption of a multi-disciplinary approach involving a range of methodologies from different areas of expertise (neurobiology, pharmacology, chemistry, netnography) all integrated to clarify some aspects of the index NPS, which were not yet available in the current literature. Additional studies are needed to better explain short and long-term effects of the index NPS, their abuse potential, and their interactions with other drugs of abuse.

As of today, synthetic cannabinoids are one of the biggest groups of new psychoactive substances.These substances can be used as substitutes for the psychoactive drug cannabis, avoiding the legalrestrictions on cannabis. Furthermore, a variety of synthetic cannabinoids are synthesized with eithersignificant or very minor structural differences, making the detection of said novel drugs hard to keepup with and is therefore of great importance to have standards which help in the identification of theintake of the parent synthetic cannabinoid. In this project, several metabolites ofsynthetic cannabinoids with indole/indazole cores with differentside chains was synthesized. The general strategy used in this project was to N-alkylate the desiredcore followed by amide coupling with L-tert-leucine methyl ester or L-Valine methyl esterhydrochloride which resulted in 8 potential synthetic cannabinoid metabolites.

This thesis describes in the form of a commentary on own original publications research on the problems of cannabinoids, ie. phytocannabinoids and some synthetic cannabinoids, their pharmacokinetics and effects. The work consists of four thematic areas: the pharmacokinetics of delta-9- tetrahydrocannabinol (THC) and cannabidiol (CBD) in rats, depending on the route of administration; THC concentration time profile in humans (after inhalation) and implications for transport safety; the pharmacokinetic profile of synthetic cannabinoids in rats; extraction and determination of phytocannabinoids in plant material. The first part of the thesis was to determine pharmacokinetic profiles of THC, CBD and combination thereof (1:1 weight ratio) in rats with respect to administration common in humans, i.e. inhalation, oral and subcutaneous administration. THC, its metabolites (11-hydroxy-tetrahydrocannabinol, 11-OH-THC; 11-nor-delta-9- carboxytetrahydrocannabinol, THCOOH) and CBD concentrations in serum and brains of animals were monitored at the 24 hours experimental interval during the study. Except for inhalation administration, co-administration of CBD inhibited THC metabolism (after both oral and subcutaneous), resulting in an increase in THC concentrations in both serum and brain of the rats relative to...

This thesis has been performed at Clinical Chemistry at Sahlgrenska University Hospital in Gothenburg. The purpose of the project was to investigate new and alternative ways to determinate synthetic cannabinoids by gas chromatography-mass spectrometry. Currently, the possibilities to quantify synthetic cannabinoids are very limited. This can lead to an increased use of synthetic cannabinoids as the risk of detection is low, which may be known by drug users. The synthetic cannabinoids are sold mixed with different herbs and have varying names like Spice Gold, Spice Silver, K2, Smoke and Pot-pourri. The synthetic cannabinoids analyzed were JWH-018 and JWH-073, which are commonly found in seized Spice material. At intake of these drugs, usually through smoking, cannabis-like effects arise. This is because they bind to cannabinoid receptors in a similar way as THC does, which is the primary active cannabinoid of cannabis. For urine samples an analytical method would probably be the most sensitive if the major metabolite could be analyzed, as it is expected to be present in high concentrations in this sample type. Since information regarding the metabolism of synthetic cannabinoids is very limited there may be reasons to analyze the mother substance in urine. Further, in plasma and serum samples the mother substance is expected in high concentrations. Thus different ways to detect JWH-018 and JWH-073 directly were investigated in this project. Derivatization of JWH-018 and JWH-073 was the first step to get more selective and sensitive GC-MS analysis. Different derivatization-reagents were investigated, for example BSTFA and TFAA. The results show that the derivatization of JWH-018 with BSTFA after reduction and extraction was successful. To achieve this, samples had to be heated at 115°C for 1-3 hours, but still the samples were not completely derivatized. The results indicate that JWH-substances are difficult to derivatized, but they are possible to derivatize with BSTFA. This could mean that a GC-MS-method maybe could be established for these substances, preferably trough TFAA-derivatization.

The aim of this study was to investigate the utility of direct analysis in real time quadrupole time-of-flight mass spectrometry and gas chromatography quadrupole time-of-flight mass spectrometry in the analysis of 162 different synthetic cannabinoids. Direct analysis in real time quadrupole time-of-flight mass spectrometry is shown to be a rapid and accurate analytical method for synthetic cannabinoids. Spectra can be generated with less than 1.5 ng of the drug in under a minute and be successfully searched against previously generated ESI-QTOF libraries in most cases (118/130 drugs tested) as well as can also be applied to the identification of synthetic cannabinoids in a mixture. Gas chromatography quadrupole time-of-flight mass spectrometry, while requiring a much longer analysis time, is shown to accurately distinguish all but 19 compounds (140/159). These two instruments have proven to be viable alternatives in synthetic cannabinoid analysis and will greatly benefit forensic laboratories.