The low dose and high dose particpants had detectable levels of CBN, THC, CBG, THC-V, and THCA-A following consumption of the joint. CBD was detected in the low dose participant immediately after smoking. THC was also detected in the placebo participant prior to smoking the joint. THCCOOH-gluc, THC-gluc, 11-OH-THC, and THCCOOH were not detected in any of the oral fluid samples tested.This work expands upon the prior knowledge of working with OF collected in the Quantisal devices and LC-MS/MS methods to simultaneously quantify ten cannabinoids. The simplistic design of this method was intentional to demonstrate feasibility in future use in driving under the influence of cannabis testing. Various parameters were optimized during method development. We evaluated multiple SPE bed volumes , with multiple combinations of washing and elution conditions . Additional LC columns evaluated included X Select HSS C18 2.5 μm beads 2.1 mm × 150 mm, XSelect HSS T3 2.5 μm beads 2.1 mm × 75 mm, and HSS PFP 1.8 μm beads 2.1 mm × 50 mm using either acidified methanol or acetonitrile based mobile phase buffers. Electrospray ionization in positive and negative ion mode was completed on each analyte. Ultimately, each parameter described in the method was selected based on largest peak area with highest signal to noise, while providing sufficient chromatographic separation. All analytes in this method had an inter-day analytical bias ± 15% with an imprecision ≤15% CV. Extraction efficiencies and matrix effects were similar to previous studies. The deuterated internal standards accounted for any extraction or matrix effects allowing for the quantification of analytes within ± 20%. Similar to Desrosiers et al., CBD-d3 was selected as the internal standard for THC-V and CBG,indoor garden table since at the time of validation no deuterated internal standards were available for these two compounds.

A quantifier to qualifier ion ratio flag was observed at 0.4 ng/mL for CBG resulting in an elevated LLOQ for CBG to 1 ng/mL. THC-d3 was employed as the deuterated internal standard for THCA-A due to the closeness in retention time. THCA-A had the lowest extraction efficiency and largest matrix effect of all the cannabinoids tested. This is likely due to the adhesiveness of this molecule to glass and plastics used throughout the procedure. We did not detect 11-OH-THC, THCCOOH, THC-gluc or THCCOOHgluc in the first three participants of each group using this method. However it does seem unlikely that glucuronidated molecules will be present above those lower limits in oral fluid, since concentrations of THC-gluc in blood following controlled cannabis smoking were < 1.1 ng/mL. Negligible amounts of THC-gluc in OF has been suggested by the lack of increased THC concentrations following glucuronidase treatment. The lack of 11-OH-THC and THCCOOH in OF collected using quantisal devices is not unexpected as other published works have measured these analytes in the 0.01–0.35 ng/mL range, which is below our LLOQ in this method. Furthermore, THCCOOH has been notoriously difficult to quantify using OF collected from the quantisal device with most methods utilizing 2-dimensional GC–MS or atmospheric pressure chemical ionization LC-MS/MS with a enzymatic hydrolysis to enrich the THCCOOH pool. The inclusion of THCCOOH in OF was suggested to confirm direct inhalation and help establish a limit to rule out passive exposure. However, due to the analytical difficulties of measuring to such a small concentration THCCOOH is likely to be only useful to rule in consumption with a negative result unable to accurately rule out. We included THC-V, CBG, and THCA-A in this method to incorporate as many available cannabinoid markers as possible since this method will be used to support pharmacokinetic and pharmacodynamics studies of marijuana use. This method differs from previous methods measuring cannabinoids in OF after solid phase extraction such that this method quantifies 10 cannabinoids, whereas others have measured 6–8 in one method.

The recreational and medicinal properties of cannabis-derived preparations have been known for centuries. The pharmacological actions of cannabis have been ascribed to its major constituent, D 9 -tetrahydrocannabinol, which binds with high affinity to specific cannabinoid receptors, named CB1 and CB2 . Both receptors belong to the super family of G protein-coupled membrane receptors, inhibit adenylate cyclase and N– and Q-type calcium channel activity and stimulate potassium channel conductance . Despite these similarities, substantial differences in the primary structures of these receptors as well as in their anatomical distribution have been reported . Although expressed throughout the body,CB1 receptors are particularly abundant in the central nervous system , where they mediate the psychotropic effects of cannabimimetic drugs . By contrast, CB2 receptors have been primarily found in immune cells, suggesting a possible contribution of this receptor sub-type to cannabino idmediated modulation of the immune response . Just as the finding of opiod receptors led in the 1970s to the discovery of a series of morphine-like chemicals in the brain — the enkephalins and the endorphins —the identification of cannabinoid receptors has prompted a vast search for their naturally occurring ligands. As a result, two endogenous substances displaying cannabinoid-like effects have been identified , arachidonoylethanolamide and 2-arachidonoylglycerol . Unlike classic neurotransmitters, anandamide and 2-AG are not stored into synaptic vesicles, but are produced upon demand through the cleavage of two distinct membrane phospholipid precursors . This reaction appears to be initiated by activation of neurotransmitter receptors, as indicated by the enhanced outflow of anandamide in rat striatum following stimulation of dopamine D2-family receptors . Similarly, application of cholinergic agonists has been shown to increase 2-AG production in the rat aorta . After its release, anandamide is inactivated by carrier-mediated transport into cells followed by intracellular hydrolysis, catalyzed by a rather non-selective amidohydrolase enzyme . 2-AG, which may be taken up by cells through the same transport system as anandamide , is hydrolyzed intracellularly into glycerol and arachidonic acid by enzyme systems that include anandamide amidohydrolase and an uncharacterized monoacylglycerol lipase .

In intact astrocytoma cells, however, the contribution of anandamide amidohydrolase to anandamide hydrolysis appears to be minor, as indicated by the ineffectiveness of amidohydrolase inhibitors to prevent 2-AG metabolism . The discovery of natural agonists at cannabinoid receptors and the identification of their biochemical pathways of formation and inactivation have spurred new interest on the physiological roles of these molecules throughout the body. These efforts have led to the identification of a possible regulatory function of the endocannabinoid system in the processing and execution of motor behaviors.The ability of cannabimimetic drugs to influence motor and cognitive performances is well documented . Indeed, cannabinoid administration in animals is accompanied by profound effects on motor behaviors , which include catalepsy, decreased motor activity and attenuation of d-amphetamine-induced hyperactivity and stereotypy . In humans, marijuana intoxication causes impaired performances in tests requiring fine psychomotor control . Moreover, cannabinoid substances produce a large spectrum of psychotropic effects, including euphoria, working memory deficits and altered perception of space and time . The psychomotor effects of cannabimimetic drugs are consistent with the anatomical distribution of CB1 receptors, which are highly expressed in areas of the CNS that play a key role in the regulation and planning of motor actions, such as the basal ganglia, cerebellum and neocortex . In keeping with this distribution, the inactivation of the CB1 receptor gene by homologous recombination produced a phenotype characterized by severe motor impairment and functional reorganization of the basal ganglia , a forebrain region involved in the sensorimotor and motivational aspects of behavior . Furthermore, in vivo microdialysis studies carried out in the rat striatum have shown the presence of extracellular levels of anandamide, which are modulated by activation of dopamine D2-family receptors . These observations not only indicate that anandamide represents a primary component of the network of neurochemicals in the striatum, but also suggest a possible cross-talk between the endocannabinoid system and other neurotransmitters regulating basal ganglia functions. Although conclusive evidence for such interactions is still lacking,microgreens grow rack neuroanatomical studies have shown that striatal CB1 receptors are mainly localized in GABA-ergic medium-spiny neurons and are co-expressed with m -opioid receptors . Moreover, it is known that exogenous administration of cannabinoids can inhibit the stimulation-evoked release of striatal neurotransmitters, such as g -aminobutyric acidand regulates proenkephalin mRNA levels in the striatum .There is substantial evidence supporting a role for the cannabinoid system as a modulator of dopaminergic activity in the basal ganglia. Administration of exogenous cannabinoids was found to increase dopamine release in rat nucleus accumbens and to excite dopaminergic neurons in the ventral tegmental area and substantia nigra . However, other studies indicate that cannabinoids potentiate the behavioral effects of dopamine antagonists and reduceelectrically evoked dopamine release from rat striatal slices . The possibility suggested by these results, that cannabinoids may regulate dopamine functions is supported by several biochemical and behavioral studies. In vivo experiments indicate that chronic treatment with dopamine D2-family receptor antagonists up-regulates CB1 receptor expression in the rat striatum . Further, injection of cannabinoid receptor agonists into the basal ganglia counteracts the motor responses of locally administered D2-receptor agonists . Even further, the hyperactivity associated with post-synaptic D2 receptor activation is accompanied by a dramatic increase of anandamide output in the striatum and is potentiated by the CB1 antagonist SR141716A . In keeping with these results, administration of the anandamide transport blocker, AM404 , has been shown to counteract several characteristic responses mediated by activation of post-synaptic D2-like receptors, such as apomorphine-induced yawning and quinpirole-induced motor activation . Taken together, these data point to a key role of the endogenous cannabinoid system in the regulation of psychomotor activity, and suggest that this system may offer a therapeutic target in pathologies involving a dysregulation of dopamine neurotransmission.The potential therapeutic use of cannabinoids for the treatment of psychomotor disorders is not only a matter of speculation. Indeed, pre-clinical studies have shown that blockade of CB1 receptors may be beneficial in the management of dyskinesias resulting from prolonged dopaminebased therapies in Parkinson’s disease . Furthermore, oral administration of D 9 -THC has been reported to alleviate tics and compulsive behaviors in patients affected by Tourette syndrome .

Certain similarities between cannabis intoxication and some psychotic symptoms have focussed the attention of psychiatrists on the possible involvement of cannabinoids in the pathogenesis of schizophrenia . Heavy cannabis use may precipitate a toxic psychosis in individuals with a previous history of psychotic illness . This observation has led to propose a ‘cannabinoid hypothesis of schizophrenia’, which postulates that the psychotic symptoms of this disease result from a over-activity of the endogenous cannabinoid system . In accordance with this theory, clinical trials of the CB1 receptor antagonist SR141716A, as a novel antipsychotic, are currently under way. However, down-regulation of CB1 cannabinoid receptors resulting from exposure to high levels of cannabinoid drugs may dampen the ability of the endogenous cannabinoid system to counteract dopamine actions, thus contributing to the manifestation of psychotic symptoms. This possibility is supported by the observation that chronic treatment with D2- family antagonists results in up-regulated expression of CB1 receptor mRNA in striatum , and by the finding that the behavioral responses induced by d-amphetamine — a screening test for antipsychotic drugs — are blocked by D 9 -THC administration in non-habituated animals, but are potentiated in animals made tolerant to cannabinoids . In this context, the elevated levels of anandamide found in the cerebrospinal fluid of schizophrenic patients might result from a homeostatic adjustment of the endogenous cannabinoid system to a functional hyperdopaminergia, rather than being a direct cause of psychosis. Likewise, the propensity of schizophrenic patients to consume more cannabis than normal individuals might be interpreted as a misguided attempt to ‘self-medicate’ the symptoms caused by a dysregulation of dopamine neurotransmission. Further investigations aimed at measuring CB1 receptor expression and determining the neuronal origin of the anandamide in CSF in a larger sample of patients may help elucidate the possible contribution of the endocannabinoid system to the pathogenesis of schizophrenia.Criminologists call crimes that have occurred, but that are not recorded or reported, the “dark figure of crime”, and they form a group of important missing statistics in understanding crime. Ever since crime statistics began being formally collected in the 19th century, this group of missing statistics has been a problem that has plagued law enforcement and criminologists.