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As forensic scientists, we are required to accurately test for certain substances. This may necessitate the use of presumptive tests such as the One Step Multi-Drug Screen Test Card with the Integrated iCup®/iCup®A.D . There are many circumstances where these tests are applicable, such as job-related drug testing, custody and parole cases. An immunoassay, or presumptive test, is designed to give the analyst, even a non-scientific analyst, a general idea of what substance(s) are present in the individuals system, so that he or she is able to more accurately confirm what substances, if any, the individual may have used or consumed. The goal of the validation study of the One Step Multi-Drug Screen Test Card with the Integrated iCup®/iCup®A.D was to determine the sensitivity of various THC containing compounds (delta-9-THC, 11-nor-9-carboxy-delta9-THC, and 11-hydroxy-delta-9-THC) as well as different solutions containing Synthetic Cannabinoids at various concentrations and stored at different temperatures. Each of the drugs were tested below, at and above the cut-off of the drug stated by the manufacturer. The cut-off of 11-nor-9-carboxy-delta9-THC, given by the manufacturer, was 50 ng/mL. For every trial that was conducted, the drug could be detected in the iCup® at this limit of detection of 50 ng/mL, except when the drug had been stored in the freezer for approximately two months prior to use. Delta-9-THC was given a cut-off of 15,000 ng/mL, which is a high concentration, especially when these assays are used in custodial cases and job-related drug tests, where living individuals are providing a fresh specimen. The concentrations of delta-9-THC and 11-hydroxy-delta-9-THC were higher than the cut-off for a positive result of 11-nor-9-carboxy-delta9-THC, but it was tested below the 15,000 ng/mL cut-off for delta-9-THC, established by the manufacturer. After these adjustments were made, both delta-9-THC and 11-hydroxy-delta-9-THC could be detected in a range between 1,500 ng/mL and 5,000 ng/mL. While 1,500 ng/mL is still high for a living specimen, it is substantially lower than 15,000 ng/mL. Analyzing the higher concentration of the synthetic cannabinoid working stock solution of 10,000 ng/mL, positive results were detected at 3,500 ng/mL and 5,000 ng/mL. There were eight cannabinoids, metabolites, and synthetic cannabinoids found in the working stock solution: (1) THC, (2) 11-Hyroxy-delta-9-THC, (3) 11-nor-9-Carboxy-delta-9-THC, (4) AB-FUBINACA, (5) AB-FUBINACA Met. 3, (6) AB-FUBINACA Met. 2a, (7) AB-PINACA-blood, and (8) AB-PINACA Pentatonic Acid metabolite. As the concentrations decreased, a positive result was not produced. Ultimately, the final conclusions of all the testing was that the One Step Multi-Drug Screen Test Card with the Integrated iCup®/iCup®A.D is not as sensitive when it comes to the synthetic cannabinoids, the primary compound present in marijuana (delta-9-THC), and the active metabolite of marijuana (11-hydroxy-delta-9-THC). In order to gain more accurate results using this presumptive test, the sensitivity of the iCup® for a detection of delta-9-THC and 11-hydroxy-delta-9-THC at a lower concentration should be done. By, doing this, an analyst can be more confident when deciding what confirmatory test to use based on what substances are present in a given sample.

Cannabidiol (CBD) is one of over 80 active cannabinoids found in Cannabis Sativa and is the second most abundant cannabinoid derived from the plant following d(9)-Tetrahydrocannabinol (THC). As opposed to THC, CBD does not appear to have any psychotropic effects, rather CBD is often utilized for its therapeutic properties, which include effects such as antinociception, anti-convulsion, and anti-inflammation. During the extraction of CBD from plant material, THC may be co-extracted. Therefore, screening and quantitating potential THC levels in individuals using CBD products is important in instances where the legality of use of THC does not match that of CBD. In recent years, oral fluid has gained recognition as a non-invasive and expedient matrix for both drug testing and forensic casework. Due to its relevance, oral fluid was selected for analysis. This project evaluated the detection and quantitation of CBD, THC, and two primary metabolites in oral fluid samples of a therapeutic-use cannabidiol donor using Biotage Supported Liquid Extraction (SLE) and subsequent testing by PerkinElmer QSight 220 CR LC-MS/MS in positive ionization mode All calibrators and quality controls were prepared by fortifying synthetic oral fluid with certified reference standards. Standards and samples were prepared in a 1:3 dilution with extraction buffer. Calibrators were prepared at 0.25, 0.5, 1, 5, 10, 50, 100, 200, 300, 400, and 500 ng/mL, with quality controls analyzed at 0.75, 70, and 425 ng/mL. Internal standard was added to the appropriate samples to account for any variation produced by sample preparation. SLE was performed using ISOLUTE SLE+ 1 mL columns with elution in hexane:ethyl acetate:methyl tert-butyl ether (80:10:10), followed by evaporation using an Organomation Multivap Nitrogen Evaporator (Berlin, Massachusetts). All samples were reconstituted in 100 uL of 0.1% formic acid in deionized water:0.1% formic acid in acetonitrile (70:30). Validation parameters were assessed using Academy Standards Board (ASB) Standard 036-Standard Practices for Method Validation in Forensic Toxicology, including linear dynamic range (LDR), limit of detection (LOD), limit of quantitation (LOQ), analyte recovery, ion suppression/enhancement, and carryover. Following reconstitution, samples were placed onto the autosampler for injection and subsequent chromatographic separation using a PerkinElmer Brownlee C18 2.1x50 millimeter (mm), 2.7 micrometer (um) column. Analysis of the samples by mass spectrometry was performed in positive mode with multiple reaction monitoring (MRM). Total run time including equilibration was 10.5 minutes. All compounds were quantified using linear calibration models with 1/X weighting (1/concentration) and measured values were normalized by their respective internal standards. The LDR was determined to be 0.25 to 500 ng/mL. For all analytes, LOD was assessed and determined to be 0.25 ng/mL with an LOQ of 1 ng/mL. Carryover was assessed by running a double blank following a sample spiked at 500 ng/mL with no analytes observed. The donor samples were collected at several timepoints around the oral administration of an 8 mg dose of CBD. These timepoints included: prior to administration, at the time of administration, 30 minutes post-administration, 45 minutes post-administration, 60 minutes post-administration, 90 minutes post-administration, and 120 minutes post-administration. CBD was quantified within the diluted oral fluid samples from below LOQ to 325.75 ng/mL. THC was detected above LOD but below LOQ, concentrations which lie below typical cut-offs used in both workplace drug testing and forensic casework. The two metabolites were not detected above the LOD. As such, the CBD product can be concluded to be of reasonable purity as it relates to legal implications. Overall, the use of laminar flow mass spectrometry was effective in detecting various cannabinoids in oral fluid samples following SLE sample extraction.

Indiana University-Purdue University Indianapolis (IUPUI) / Cannabinoid receptors (CBRs) are inhibitory G-protein coupled receptors (GPCRs) that bind endogenous and exogenous cannabinoids. In an unaltered state, endogenous cannabinoids regulate system function and synchrony. Administration of cannabinoids such as Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD), which are found in the cannabis plant, can lead to disruptions in well-maintained inhibitory signaling. Although marijuana usage rates have been relatively stable since 2002, the number of young adolescents and adults that report perceiving marijuana as a “no risk” drug has doubled to more than 17% in each age group (Azofeifa et al., 2016). However, no drug is fully without risks. Preclinical studies have indicated that a history of THC during adolescence, but not adulthood, results in object memory impairments following a period of no-drug administration. In tests of unconditioned anxiety, acute THC evokes anxiety-like activity at higher doses. Conversely, CBD blocks object memory impairment in models that produce inflammation and also produces anxiolytic activity. Although THC and CBD are often used together for recreational and medical purposes, no study has observed the acute and long-lasting effects of THC+CBD in a battery of tests. The current work sought to fulfill three specific aims of research to identify both age and sex differences in response to cannabinoids. In Aim 1, a dose-response to acute THC or CBD was assessed in male and female adolescent and adult mice on the elevated plus maze (EPM) and open field (OF) activity. In Aim 2, acute vehicle, 10 mg/kg THC, 20 mg/kg CBD, and THC+CBD were assessed for their effects on memory consolidation, EPM, and OF activity in male and female mice during adolescence or adulthood. Mice from Aim 2 received a total of 8 injections over a 3 week period, then were given 3 weeks of rest. In Aim 3, all mice were tested again for object memory, EPM, and OF activity under no-drug conditions to assess the effects of an adolescent or adult history of cannabinoids in male and female mice. Results of Aim 1 indicated that adult mice, regardless of sex, were more sensitive to the acute effects of THC on unconditioned anxiety and locomotor activity. A rapid tolerance to THC may develop, as mice tested on the EPM in Aim 2 following their second injection of THC did not demonstrate anxiety-like activity that was present in Aim 1. However, anxiety-like activity persisted in the open field, and acute administration of THC+CBD resulted in synergistic effects on anxiety in adult females over THC alone. Interestingly, acute THC in adolescent males rescued a deficit in object memory in the vehicle group, whereas only adult males receiving vehicle showed significant object discrimination. Females were relatively resistant to effects of acute cannabinoids on object memory, with adolescents being completely insensitive. Results of Aim 3 indicated minimal effects of a history of cannabinoids on behavior. In contrast to previous experiments, an adolescent history of THC did not impair object memory. Interestingly, females administered THC+CBD during adulthood demonstrated impaired object memory following a no-drug period. Although CBD is often considered to lack a psychoactive profile, it is hypothesized that this impairment may be due to actions of CBD on 5HT1a receptors and require a fully-developed stress and gonadal system. The current results indicate that acute cannabinoid administration results in anxiety-like behavior when administered during adulthood, and that an adult history of THC+CBD in females results in impaired cognitive behavior. As the effects of cannabinoids were primarily present in adults, this may suggest that the fully-developed brain is more susceptible to interruption by acute and repeated exogenous cannabinoid administration and that adolescents may have a higher threshold for impairment of behavior.

Indiana University-Purdue University Indianapolis (IUPUI) / Cannabis (and its main psychoactive component, THC) is one of the most widely-used drugs in the world, and recent expansion of its legal status has made it available in a variety of formulations and at a potency unrivaled in history. While its medicinal properties are gaining scientific support, so too is its potential to lead to abuse and dependence. Both initiation of cannabis use and frequent cannabis use are most prevalent in adolescence, and compared to adults, cannabis use by adolescents is associated with a greater likelihood of developing cannabis dependence and cannabis use disorder. Given the ethical limitations surrounding research that provides cannabis to non-users or non-adults, animal models of drug use can be valuable tools for the study of causes and consequences related to drug use, as well as allowing for investigating brain mechanisms underlying these factors. However, only recently have models in which animals reliably use cannabis (THC) at levels above its respective vehicle and at levels which produce consistent behavioral and physiological effects become available, and in no case has age-related differences in this use been examined. Thus, one goal of the current study was to directly compare the self-administration of edible THC (a route of administration used by humans and a formulation increasing in popularity) between adolescent and adult mice. Adolescents also appear to be differentially sensitive to various effects of several classes of drugs, and they have been shown to be less sensitive to the aversive effects of cannabis, thereby putting them at greater risk for elevated and continued use. Evidence also suggests that, in addition to the risk associated with adolescent cannabis use, having initial positive subjective experiences resulting from its use is a strong predictor of subsequent cannabis dependence. Thus, the second goal of the current study was to use the place conditioning paradigm to examine the reward- (or aversion-) inducing properties of THC in adolescent and adult C57BL/6J mice, using both the traditional experimenter-administered THC (via injection) as well as edible THC self-administration. Prior to initiating these THC studies, sensitivity of the place conditioning procedure to age-related differences in drug-induced reward was validated using cocaine, yielding locomotor stimulation in both ages and a decreased sensitivity to cocaine’s rewarding properties in adolescent mice. When provided limited access to edible THC dough in doses ranging from 0.0 to 6.0 mg/kg, mice showed a dose-dependent reduction in consumption across access sessions, and this reduction was more rapid in adult mice at the highest doses, suggesting that adolescent mice might have been less sensitive to its aversive properties. These same mice, as well as a separate group of mice receiving injection (also 0.0 to 6.0 mg/kg THC), were given place conditioning sessions, alternating between THC dough and control dough or THC injection and vehicle injection, for 6 days per week and were tested once per week across a total of 3 weeks. Mice conditioned using edible THC showed a neutral response (neither reward nor aversion) at all doses. However, mice conditioned using injected THC showed a conditioned place aversion to the highest dose, which was more pronounced in adult mice. Interestingly, in mice self-administering edible THC, the dose of THC consumed was related to the outcome of place conditioning, such that a conditioned place preference was observed for adult mice which shifted their consumption of 3.0 mg/kg edible THC downward relative to those mice with full consumption of 3.0 mg/kg, and for adolescent mice which had the highest degree of consumption of 6.0 mg/kg edible THC relative to those mice with the lowest consumption of 6.0 mg/kg. Furthermore, initial place preference outcomes at the individual level at test 1 predicted subsequent doses of edible THC consumed, suggesting mice adjust their self-administration of edible THC based on the subjective experience it produces. Besides its impact in place conditioning, THC also had differential effects on body weight and locomotor activity based on age and route of administration. Collectively, this project demonstrates that adolescent mice are less sensitive to the hedonic properties of both cocaine and THC, and that differences in edible THC self-administration between ages, and between individuals within an age, are likely related the subjective experience of its rewarding and aversive properties.

Drug screening is widespread in contexts such as the criminal justice system, employment, and substance abuse treatment centers. Traditionally, drug testing methods have targeted urine matrices and extensive research is available regarding urine drug screening. Due to the nature of sample collection, urine specimen may be tampered or adulterated in efforts to invalidate or pass a drug test. Examples of this include substituting a sample with synthetic urine, diluting a sample with water, or adding a substance to the sample. The addition of a substance is referred to as adulteration and is done in an effort to mask drugs in the sample or to invalidate the test results. For various reasons, including the effects of adulteration, time, and costs associated with urine drug tests, oral fluid (OF) has become an increasingly important alternative matrix for screening drugs of abuse. It offers distinct advantages since tests can be administered noninvasively, quickly, and under observation, thus reducing the risk of tampering. Studies have also shown that drugs of abuse detected in OF may correlate better to user impairment at the time of collection, as compared to other matrices. In 2019, the Substance Abuse and Mental Health Service Administration (SAMHSA) published mandatory guidelines for oral fluid testing. Although these guidelines only directly impact federal spaces, they also serve as a blueprint for developing drug testing laws and policies in other organizations. Despite requirements and procedures controlling for specimen adulteration, it is recognized that manufacturers will continue to develop and market new products to avoid drug detection, just as with urine drug tests. The design of this experiment was to investigate the effects of the commercially available drug testing subversion products High Voltage Saliva Cleanse Mouthwash (High Voltage Detox, Las Vegas, Nevada, USA) and Stinger Detox Mouthwash (Stinger Detox, Phoenix, Arizona, USA) on immunoassay testing for tetrahydrocannabinol (THC) and methamphetamine (MET) in OF. The High Voltage Saliva Cleanse was designated adulterant “A”, and the Stinger Detox was designated adulterant “B”. Two separate immunoassay kits, Discover™ (American Screening Corporation, Inc., Shreveport, Louisiana, USA) and Orawell® (Jiangsu Well Biotech Co., Ltd, Changzhou, Jiangsu, China), were assessed to investigate the differences in performance of the current available test devices in addition to the effects of the subversion products. Using Discover™, samples were spiked according to 0.5 times (x), 1x, and 2x the cutoff concentrations of 50 ng/mL THC and 50 ng/mL MET without adulterant, with Adulterant A, and with Adulterant B. Testing with Orawell® devices was initially conducted at 1x and 2x the cutoff concentrations of 40 ng/mL THC and 50 ng/mL of MET. Based on the lack of response, testing at 0.5x was not conducted. Additional testing was conducted at 1.5x and 3x the cutoff concentrations without adulterant, with Adulterant A, and with Adulterant B. It was concluded that the adulterants affected the test results in the Orawell® device, by producing false positives for drugs of abuse not present in the sample for 17 (56.7%) of the 30 tests containing adulterants. Additionally, it was concluded that both immunoassay tests assessed were lacking in analytical sensitivity and reproducibility.

The popularity of cannabis and reduction of cannabis prohibition in the United States has led to increased consumption in human users. However, relatively little is understood about the abuse potential of cannabis and its main psychoactive ingredient, THC. One reason for the lack of insight into the addictive effects of THC is that the animal models investigating voluntary intake of THC have been hampered by low rates of behavior and THC intake often does not surpass intake of vehicle. We hypothesized that, in addition to supporting operant behavior (smoking, vaping, or consuming edibles) THC might increase the reinforcing effects of non-drug rewards (e.g., playing video games, listening to music, eating snacks). To investigate this hypothesis, we evaluated whether THC injections could increase responding for saccharin (0.2% w/v, SACC) in male and female rats. During our investigation we noted that the pharmacology of THC was complex, with potent motor suppressant effects, and that changes in behavior depended on the pharmacokinetics of THC administration. To further explore the pharmacokinetics, we conducted 3 experiments that manipulated THC dose (Experiment 1), Injection-Test interval (Experiment 2) and Injection-Injection interval (washout duration, Experiment 3). We hypothesized that THC would increase responding for SACC, but that this effect would depend on having a longer time between sessions to reduce motor-suppressing effects of THC accumulation. Male and female rats were shaped to respond for SACC under a progressive ratio (PR) schedule of reinforcement. The PR schedule measures motivation by increasing the response requirement after each reinforcer is earned. In Experiment 1 there was a significant effect of THC dose, with moderate doses (0.3-0.75 mg/kg) increasing motivation for SACC and high doses (3 mg/kg) causing significant motor suppression. In Experiment 2 (Injection-Test interval) we found that the timing of THC injections was critical – enhancing effects were observed soon after THC injections were administered (30-60 min) but after 120 min THC no longer increased motivation for SACC. Finally, in Experiment 3 (Injection-Injection interval) we found that daily injections of THC (24 h washout) resulted in significant decreases in motivation from Session 1 to session 8. In contrast, 72 h washout intervals resulted in stable enhancement of motivation for SACC by THC. These studies indicate that the reinforcement enhancing effects of THC are robust but depend critically on the pharmacokinetics and bioavailability of THC.

Humans consume cannabis for the pharmacological effects mediated by the primary psychoactive cannabinoid, delta-9-tetrahydrocannabinol (THC). However, there is little evidence to suggest that THC acts as a primary reinforcer in non-human models because the drug alone does not support robust self-administration. We hypothesized that THC may have more potent reinforcement enhancing effects – meaning that THC may enhance the reinforcing effects of other non-drug rewards in a user’s environment. In the present experiments, we explore the effects of THC on operant responding for saccharin (SACC) or a visual stimulus (VS). In all experiments rats were shaped to respond for their assigned reinforcer. Drug challenge tests were conducted every 72 hours, rats were injected with the assigned dose of THC and responding for each reinforcer was measured. Our initial findings indicated possible sex differences between male and female rats – THC injections increased lever-pressing for SACC in male rats but not female rats. However, in follow-up experiments we used a different response (nose-key press instead of lever press) that facilitated operant responding in rats that were different sizes – adult males are significantly more massive than adult females. In that experiment THC enhanced nose-key presses for SACC in both male and female rats across a range of doses. Moreover, this latter experiment confirmed that the effect of THC was motivational in nature, THC injections increased effort to obtain SACC under a progressively increasing schedule of reinforcement (progressive ratio). Finally, using a third operant response (head entry into a receptacle) we demonstrated that THC increased reinforcement by the VS across a range of doses. The present studies indicate that THC acts as a reinforcement enhancer, increasing motivation in male and female rats to obtain both SACC and VS throughout a range of doses. By demonstrating that THC enhances the reinforcing effects of both gustatory and non-gustatory reinforcers, our evidence supports the hypothesis that THC’s effect on the brain facilitates incentive motivation regardless of sensory modality of the reinforcer.

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Objectives: Both substance use, on the one hand, and the first signs of psychosis, on the other, commonly begin in adolescence. Adolescents with substance use disorder (SUD) frequently show recreational use of cannabis and 3,4-methylenedioxymethamphetamine (MDMA). When attenuated psychotic symptoms (APS) occur during the course of SUD, they are commonly attributed to the cannabis use, neglecting the role of other substances abused, such as MDMA in the risk of psychosis. Methods: We analyzed retrospective self-reports on APS (Prodromal Questionnaire, PQ-16) and amount of cannabis and MDMA use in n = 46 adolescent psychiatry outpatients with SUD. N = 17 (35%) individuals reported MDMA consume additional to cannabis. Furthermore, we examined the associations of APS with cannabis and MDMA use in stepwise hierarchical regressions while controlling for trauma history, birth complications and gender. Results: APS were not related to cannabis (B = 0.04, p = 0.842), but to MDMA use (B = 4.88, p = 0.001) and trauma history (B = 0.72, p = 0.001). Gender (B = −0.22, p = 0.767) and birth complications (B = −0.68, p = 0.178) were not associated with APS. Discussion: Our results indicate that MDMA use additional to cannabis use is associated with APS among adolescent SUD patients. Contrary to our expectations, we did not see an association of cannabis use and APS. We speculate that cannabis increases the risk for psychosis after a longer period of use and in combination with other risk factors, such as trauma history. Clinicians should screen for APS among SUD patients using MDMA and cannabis in order to adapt treatment plans of SUDs. Future research should validate these findings in longitudinal studies including polysubstance use and trauma history.