Influence of Cannabinoid Receptor 1 Genetic Variants on the Subjective Effects of Smoked Cannabis.

As many jurisdictions consider relaxing cannabis legislation and usage is increasing in North America and other parts of the world, there is a need to explore the possible genetic differences underlying the subjective effects of cannabis. This pilot study investigated specific genetic variations within the cannabinoid receptor 1 (CNR1) gene for association with the subjective effects of smoked cannabis. Data were obtained from a double-blinded, placebo-controlled clinical trial studying the impact of cannabis intoxication on driving performance. Participants randomized to the active cannabis group who consented to secondary genetic analysis (n = 52) were genotyped at the CNR1 rs1049353 and rs2023239 polymorphic areas. Maximum value and area under the curve (AUC) analyses were performed on subjective measures data. Analysis of subjective effects by genotype uncovered a global trend towards greater subjective effects for rs1049353 T-allele- and rs2023239 C-allele-carrying subjects. However, significant differences attributed to allelic identity were only documented for a subset of subjective effects. Our findings suggest that rs1049353 and rs2023239 minor allele carriers experience augmented subjective effects during acute cannabis intoxication.

Cannabis constituents interact at the drug efflux pump BCRP to markedly increase plasma cannabidiolic acid concentrations.

Cannabis is a complex mixture of hundreds of bioactive molecules. This provides the potential for pharmacological interactions between cannabis constituents, a phenomenon referred to as "the entourage effect" by the medicinal cannabis community. We hypothesize that pharmacokinetic interactions between cannabis constituents could substantially alter systemic cannabinoid concentrations. To address this hypothesis we compared pharmacokinetic parameters of cannabinoids administered orally in a cannabis extract to those administered as individual cannabinoids at equivalent doses in mice. Astonishingly, plasma cannabidiolic acid (CBDA) concentrations were 14-times higher following administration in the cannabis extract than when administered as a single molecule. In vitro transwell assays identified CBDA as a substrate of the drug efflux transporter breast cancer resistance protein (BCRP), and that cannabigerol and Δ9-tetrahydrocannabinol inhibited the BCRP-mediated transport of CBDA. Such a cannabinoid-cannabinoid interaction at BCRP transporters located in the intestine would inhibit efflux of CBDA, thus resulting in increased plasma concentrations. Our results suggest that cannabis extracts provide a natural vehicle to substantially enhance plasma CBDA concentrations. Moreover, CBDA might have a more significant contribution to the pharmacological effects of orally administered cannabis extracts than previously thought.

Determination of in vitro human whole blood-to-plasma ratio of THJ-018 utilizing gas chromatography-Mass spectrometry.

OBJECTIVES: This study was aimed to determine in vitro human whole blood-to-plasma ratio (KWB/P) of THJ-018 by gas chromatography/mass spectrometry (GC/MS). MATERIALS AND METHODS: The samples (human blood) were sprayed with THJ-018 and an internal standard and extracted using solid-phase extraction. THJ-018 was determined in the final extracts by GC/MS. RESULTS: The value for KWB/P was 1.56 (1.38-1.81), and red blood cell partitioning was 1.01 (1.01-1.02). The distribution of THJ-018 between whole blood and plasma was observed to be affected by temperature. CONCLUSION: The data analysis supports the proposition that the ratio of the plasma to whole blood concentrations (1.56) is a suitable parameter characterizing THJ-018 distribution in whole blood. For toxicological analysis, it would be best to refrain from converting any drug concentration measured in whole blood to that anticipated in plasma or serum; however, toxic and therapeutic concentrations should be determined for the individual specimens collected.

Intoxication by a synthetic cannabinoid (JWH-018) causes cognitive and psychomotor impairment in recreational cannabis users.

BACKGROUND: Smoking mixtures containing synthetic cannabinoids (SCs) have become very popular over the last years but pose a serious risk for public health. Limited knowledge is, however, available regarding the acute effects of SCs on cognition and psychomotor performance. Earlier we demonstrated signs of impairment in healthy volunteers after administering one of the first SCs, JWH-018, even though subjective intoxication was low. In the current study, we aimed to investigate the acute effects of JWH-018 on several cognitive and psychomotor tasks in participants who are demonstrating representative levels of acute intoxication. METHODS: 24 healthy cannabis-experienced participants took part in this placebo-controlled, cross-over study. Participants inhaled the vapor of 75 µg JWH-018/kg body weight and were given a booster dose if needed to induce a minimum level of subjective high. They were subsequently monitored for 4 h, during which psychomotor and cognitive performance, vital signs, and subjective experience were measured, and serum concentrations were determined. RESULTS: Maximum subjective high (average 64%) was reached 30 min after administration of JWH-018, while the maximum blood concentration was shown after 5 min (8 ng/mL). JWH-018 impaired motor coordination (CTT), attention (DAT and SST), memory (SMT), it lowered speed-accuracy efficiency (MFFT) and slowed down response speed (DAT). CONCLUSION: In accordance with our previous studies, we demonstrated acute psychomotor and cognitive effects of a relatively low dose of JWH-018.

Optimization of cloned enzyme donor immunoassay cut-offs for drugs of abuse in post-mortem whole blood.

INTRODUCTION: Immunoassay (IA) tests are not widely applied in post-mortem samples, since they are based on technologies requiring relatively non-viscous specimens, and compounds originating from the degradation of proteins and lipids during the post-mortem interval can alter the efficiency of the test. However, since the extraction techniques for IA tests are normally rapid and low-cost, IA could be used as near-body drug-screening for the classes of drugs most commonly found in Italy and Europe. In this study, semi-quantitative results on post-mortem whole blood samples obtained through CEDIA analysis (cannabinoids, cocaine, amphetamine compounds, opiates and methadone), were compared with results of confirmatory analysis obtained using GC-MS. Screening cut-offs for all drugs were retrospectively optimized. METHODS: Post-mortem whole blood samples from autopsy cases of suspected fatal intoxication were collected over 3 years. Samples were initially analyzed through CEDIA (CEDIA, ILab 650, Werfen). Confirmatory analyses were then performed by GC-MS (QP 2010 Plus, Shimadzu). Screening cut-offs were retrospectively optimized using Receiver Operating Characteristic (ROC) analysis. RESULTS: CEDIA results were available for 125 samples. Two-hundred-eighty-nine (289) positive screening results were found. Among these, 162 positive confirmation results were obtained. Optimized screening cut-offs were as follows: 6.5ng/ml for THC; 4.2ng/ml for THC-COOH; 12.0ng/ml for cocaine; 6.6ng/ml for benzoylecgonine; 6.4ng/ml for opiates; 2.0ng/ml for methadone. Analysis of ROC-curves showed a satisfying degree of separation in all tests except for amphetamine compounds, with areas under the curve (AUC) between 0.915 (THC) and 0.999 (for benzoylecgonine and methadone). DISCUSSION: The results of the study showed that CEDIA screening at the optimized cut-offs exhibits a very high sensitivity and good specificity and positive predictive value (PPV) for cannabinoids, cocaine and metabolites, opiates and methadone. A high number of false positives (n=19) for amphetamine compounds was observed at the optimized cut-off, resulting in a very low PPV, which is also influenced by the very low number of TP (n=4). CONCLUSION: The results of the study show that the CEDIA is a valuable screening test on post-mortem whole blood for cannabinoids, cocaine and metabolites, opiates and methadone, but it is not recommended for amphetamine compounds, due to the high number of false positives. The strengths of the study are the large sample size, the inclusion of post-mortem cases only and the high level of sensitivity and specificity obtained at the optimized cut-offs.

Parallel Reaction Monitoring-Based Quantification of Cannabinoids in Whole Blood.

Cannabis is the most consumed drug of abuse, making it the primary target for identification and quantification in human whole blood regarding forensic and clinical toxicology analyses. Among biological matrices, blood is the reference for toxicological interpretation. A highly sensitive and selective liquid chromatography (LC) hyphenated with high-resolution mass spectrometry (HRMS) was developed for the quantification of Δ9-tetrahydrocannabinol (THC), 11-hydroxytetrahydrocannabinol (THC-OH), 11-nor-9-carboxy-tetrahydrocannabinol (THC-COOH) and cannabidiol (CBD). Those cannabinoids were extracted from 1 mL of whole blood by a simple liquid-liquid extraction (LLE) in acidic conditions. HRMS was performed on an Orbitrap-based instrument using its trapping capabilities and increased selectivity for parallel reaction monitoring (PRM) quantification in positive polarity with a negative polarity switching for THC-OH and THC-COOH. Although selected reaction monitoring (SRM) and PRM-targeted methods have similar performance in terms of linearity, dynamic range, precision and repeatability, Orbitrap-based PRM provides a higher specificity due to the use of high-resolution mode separating background ions from the targeted molecules. The method was fully validated according to guidelines set forth by the "Société Française des Sciences et des Techniques Pharmaceutiques" (SFSTP). Trueness was measured below 107% for all tested concentrations. Repeatability and intermediate precision were found to be lower than 12% while the assay was found to be linear in the concentration range of 0.4-20 ng/mL for THC, THC-OH and CBD and of 2-100 ng/mL for THC-COOH. Recovery (RE) and matrix effect (ME) ranged from 70.6 to 102.5% and from -40 to 6.6%, respectively. The validated method provides an efficient procedure for the simultaneous and rapid quantification of cannabinoids in PRM mode providing an alternative over classical SRM.

Validation of a liquid chromatography tandem mass spectrometry (LC-MS/MS) method to detect cannabinoids in whole blood and breath.

Background The widespread availability of cannabis raises concerns regarding its effect on driving performance and operation of complex equipment. Currently, there are no established safe driving limits regarding ∆9-tetrahydrocannabinol (THC) concentrations in blood or breath. Daily cannabis users build up a large body burden of THC with residual excretion for days or weeks after the start of abstinence. Therefore, it is critical to have a sensitive and specific analytical assay that quantifies THC, the main psychoactive component of cannabis, and multiple metabolites to improve interpretation of cannabinoids in blood; some analytes may indicate recent use. Methods A liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed to quantify THC, cannabinol (CBN), cannabidiol (CBD), 11-hydroxy-THC (11-OH-THC), (±)-11-nor-9-carboxy-Δ9-THC (THCCOOH), (+)-11-nor-Δ9-THC-9-carboxylic acid glucuronide (THCCOOH-gluc), cannabigerol (CBG), and tetrahydrocannabivarin (THCV) in whole blood (WB). WB samples were prepared by solid-phase extraction (SPE) and quantified by LC-MS/MS. A rapid and simple method involving methanol elution of THC in breath collected in SensAbues® devices was optimized. Results Lower limits of quantification ranged from 0.5 to 2 µg/L in WB. An LLOQ of 80 pg/pad was achieved for THC concentrations in breath. Calibration curves were linear (R2>0.995) with calibrator concentrations within ±15% of their target and quality control (QC) bias and imprecision ≤15%. No major matrix effects or drug interferences were observed. Conclusions The methods were robust and adequately quantified cannabinoids in biological blood and breath samples. These methods will be used to identify cannabinoid concentrations in an upcoming study of the effects of cannabis on driving.

Identification of a thermal degradation product of CUMYL-PEGACLONE and its detection in biological samples.

The structural diversity of synthetic cannabinoids makes it a challenging task to have a comprehensive screening method for this class of drugs. The difficulty is increased by the fact that some synthetic cannabinoids undergo thermal decomposition during common routes of administration, such as smoking or vaping. CUMYL-PEGACLONE is a relatively new synthetic cannabinoid which has a structural variant from most other synthetic cannabinoids: a γ-carbolinone core. To investigate its thermal stability, CUMYL-PEGACLONE was heated in an oven at temperatures ranging from 200 to 350o C, and a major thermal degradation product, N-pentyl-γ-carbolinone, was subsequently identified. Unlike some other synthetic cannabinoids, the thermal degradation product of CUMYL-PEGACLONE is not one of its known metabolites, nor were any known metabolites detected during the thermal stability experiments. The degradation product was formed in significant amounts at temperatures above 250°C, and has been detected (along with CUMYL-PEGACLONE) in case samples, including post-mortem blood and urine, and residue found at a scene.

Detectability of various cannabinoids in plasma samples of cannabis users: Indicators of recent cannabis use?

To assess the informative value of determining (minor) cannabinoids in plasma of cannabis users, detection rates of 14 cannabinoids (next to Δ9 -THC and THC-COOH) were determined. 11-OH-THC, THCA, CBC, CBN, and CBD were the most frequent detectable cannabinoids. The dependency of cannabinoid detectability on the plasma Δ9 -THC concentration was examined.

Use of gas chromatography-mass spectrometry for definitive diagnosis of synthetic cannabinoid toxicity in a dog.

OBJECTIVE: To report the use of gas chromatography-mass spectrometry to confirm a diagnosis of synthetic cannabis toxicosis in a dog and to describe the clinical course of the intoxication. CASE SUMMARY: An 11-year-old neutered female Boxer dog was referred due to acute onset of vomiting, ataxia, dull mentation, and delirium that progressed to generalized seizures, unresponsive to diazepam. Prior to presentation, the dog was found lying down, minimally responsive with vomitus around it. A chewed bag containing dried plant material was found next to the dog. The dog was anesthetized and ventilated with positive pressure for 16 hours, and eventually made a full recovery. Gas chromatography-mass spectrometry analysis of the plant material and a plasma sample from the dog revealed presence of the synthetic cannabinoid N-[(1S)-1-(aminocarbonyl)-2-methylpropyl]-1-(cyclohexylmethyl)-1H-indazole-3-carboxamide, also known as AB-CHMINACA, a relatively new illegal synthetic cannabinoid, known by the local forensic police department as a drug of recreational abuse. NEW OR UNIQUE INFORMATION PROVIDED: Reports of synthetic cannabinoid toxicosis in dogs are scarce and are based on urine test kits for tetrahydrocannabinol that have not been validated in the veterinary literature. This is the first report to describe utilization of gas chromatography-mass spectrometry on canine plasma to reach a definitive diagnosis.

Quantitation of Cannabinoids in Breath Samples Using a Novel Derivatization LC-MS/MS Assay with Ultra-High Sensitivity.

As the legalization of medical and recreational marijuana use expands, measurement of tetrahydrocannabinol (THC) in human breath has become an area of interest. The presence and concentration of cannabinoids in breath have been shown to correlate with recent marijuana use and may be correlated with impairment. Given the low concentration of THC in human breath, sensitive analytical methods are required to further evaluate its utility and window of detection. This paper describes a novel derivatization method based on an azo coupling reaction that significantly increases the ionization efficiency of cannabinoids for LC-MS/MS analysis. This derivatization reaction allows for a direct derivatization reaction with neat samples and does not require further sample clean-up after derivatization, thus facilitating an easy and rapid "derivatize & shoot" sample preparation. The derivatization assay allowed for limits of quantitation (LOQ's) in the sub-pg/mL to pg/mL range for the five cannabinoids in breath samples, i.e., only 5~50 femtograms of an analyte was required for quantitation in a single analysis. This ultrahigh sensitivity allowed for the quantitation of cannabinoids in all breath samples collected within 3 hours of smoking cannabis (n = 180). A linear correlation between THC and cannabinol (CBN) in human breath was observed, supporting the hypothesis that CBN is converted from THC during the combustion of cannabis. The derivatization method was also applied to the analysis of cannabinoids in whole blood samples, achieving LOQ's at ten-pg/mL to sub-ng/mL level. This azo coupling-based derivatization approach provided the needed analytical sensitivity for the analysis of THC in human breath samples using LC-MS/MS and could be a valuable tool for the analysis of other aromatic compounds in the future.

A case of 5F-ADB / FUB-AMB abuse: Drug-induced or drug-related death?

Synthetic cannabinoids (SCs) belong to the group of new psychoactive substances (NPS) which appear sprayed on herbal mixtures on the "street" drug market and are intended for smoking like marijuana. In the present report we discuss a fatal case of 18-years-old boy, who had smoked SCs since several months and an overuse of SCs during last 48 h of his life has been apprised. The autopsy findings revealed acute respiratory distress syndrome (ARDS). Both toxicological analysis of deceased blood and urine samples and chemical analysis of the herbal mixture seized revealed presence of two SCs - 5F-ADB and FUB-AMB. The amount of 5F-ADB in blood was found to be 3.7 ng/mL by standard addition method. Severe and irreversible morphology changes in lung specimen, leading to ischemic damage of all internal organs and tissues, were observed during histological examination. The present case can be discussed as an example of both drug-induced and drug-related death resulting from acute intoxication with 5F-ADB and FUB-AMB as well as from systematic use of both synthetic cannabinoids.

First application of atmospheric-pressure chemical ionization gas chromatography tandem mass spectrometry to the determination of cannabinoids in serum.

The analysis of cannabinoids in blood samples is still a challenging issue for forensic laboratories, because of the low concentrations to be determined to prove that a person acted under CannabisTherefore, sensitive analytical techniques are required. This study presents the development and validation of a novel APGC-MS/MS method for the simultaneous determination of Δ9-tetrahydrocannabinol (THC), 11-hydroxy- Δ9-THC (THC-OH), 11-nor-9-carboxy- Δ9-THC (THCA), cannabidiol (CBD), cannabidiol acid (CBDA) and cannabigerol (CBG) in human serum. The developed method was fully validated according to international guidelines, with evaluation of selectivity, precision, accuracy, linearity, LODs and LOQs, extraction recovery and matrix effect. The method was linear in the range 0.2-25 ng/mL for THC, THC-OH, CBD and CBG, while for THCA and CBDA linearity was assessed in the range of 0.8-100 ng/mL and 3-100 ng/mL, respectively. The LOQs were determined in 0.2 ng/mL for THC, 0.4 ng/mL for THC-OH, 0.8 ng/mL for CBD and CBG, 1.6 ng/mL for THCA and 3 ng/mL for CBDA. The method was applied to the analysis of 15 serum samples from DUID cases. To the best of our knowledge, the present work is the first one describing an application of APGC source in the field of forensic toxicology.

Cannabinoid concentrations in blood and urine after smoking cannabidiol joints.

In Switzerland, the sale of cannabis with tetrahydrocannabinol (THC) content less than 1% has recently been legalized. As a consequence, cannabis with low THC and high cannabidiol (CBD) values up to approximately 25% is legally available on the market. In this study, we investigated cannabinoid blood and urine concentrations of a naive user and of a modeled chronic user after smoking a single CBD joint. Chronic use was modeled as smoking 2 joints per day for 10 days. Joints contained 200mg of cannabis with THC concentrations of 0.94% and 0.8% and CBD concentrations of 23.5% and 17% in the naive-smoker and chronic-smoker experiment, respectively. After smoking, blood and urine samples were collected for 4 and 20h after smoking start, respectively. THC blood concentrations reached 2.7 and 4.5ng/mL in the naive and chronic user, respectively. In both cases, the blood THC concentration is significantly above the Swiss road traffic threshold of 1.5ng/mL. Consequently, the user was legally unfit to drive directly after smoking. CBD blood concentrations of 45.7 and 82.6ng/mL were reached for the naive and chronic user, respectively. During the 10-day smoking period, blood and urine samples were regularly collected. No accumulation of any cannabinoid was found in the blood during this time. Urinary 11-nor-9-carboxy-THC concentrations seemed to increase during the 10-day period, which is important in abstinence testing.

Methodology for controlled administration of smoked synthetic cannabinoids JWH-018 and JWH-073.

Synthetic cannabinoids (SCs) are a significant public health concern given their widespread use and severe effects associated with intoxication. However, there is a paucity of controlled human studies investigating the behavioral and physiological effects and pharmacokinetics of these compounds. Designing a reliable method to administer consistent, concentration-dependent synthetic cannabinoids is an integral component of controlled study of these compounds. Further, optimizing methods to assess the parent compounds and metabolites in plasma is critical in order to be able to establish their pharmacokinetics after administration. To develop a reliable method to administer smokable, concentration-dependent SCs, cigarettes were prepared with plant matter adulterated with increasing concentrations of the first generation cannabinoids found in SC products, JWH-018 and JWH-073. Cigarettes were assessed 1-6 months after preparation using ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to determine compound stability over time and concentration consistency throughout the cigarettes. Optimal conditions to detect metabolites in human plasma as a function of storage temperature (-4 °C to -80 °C) and time (24 h - 1 month) were also determined. Analyses verified that the method utilized to develop SC cigarettes yielded consistent, concentration-dependent products within 25% of the expected concentrations. JWH-018, JWH-073 and metabolites in spiked plasma were stable under the time and temperature conditions; concentrations were within ±20% of target values. These studies provide techniques and methods to conduct controlled investigations of the dose-dependent effects of first generation SCs to begin understanding risks associated with use. This article is part of the Special Issue entitled 'Designer Drugs and Legal Highs.'

Oral administration of cannabis with lipids leads to high levels of cannabinoids in the intestinal lymphatic system and prominent immunomodulation.

Cannabidiol (CBD) and ∆9-tetrahydrocannabinol (THC) have well documented immunomodulatory effects in vitro, but not following oral administration in humans. Here we show that oral co-administration of cannabinoids with lipids can substantially increase their intestinal lymphatic transport in rats. CBD concentrations in the lymph were 250-fold higher than in plasma, while THC concentrations in the lymph were 100-fold higher than in plasma. Since cannabinoids are currently in clinical use for the treatment of spasticity in multiple sclerosis (MS) patients and to alleviate nausea and vomiting associated with chemotherapy in cancer patients, lymphocytes from those patients were used to assess the immunomodulatory effects of cannabinoids. The levels of cannabinoids recovered in the intestinal lymphatic system, but not in plasma, were substantially above the immunomodulatory threshold in murine and human lymphocytes. CBD showed higher immunosuppressive effects than THC. Moreover, immune cells from MS patients were more susceptible to the immunosuppressive effects of cannabinoids than those from healthy volunteers or cancer patients. Therefore, administering cannabinoids with a high-fat meal or in lipid-based formulations has the potential to be a therapeutic approach to improve the treatment of MS, or indeed other autoimmune disorders. However, intestinal lymphatic transport of cannabinoids in immunocompromised patients requires caution.

Determination of ∆-9-Tetrahydrocannabinol (THC), 11-hydroxy-THC, 11-nor-9-carboxy-THC and Cannabidiol in Human Plasma using Gas Chromatography-Tandem Mass Spectrometry.

Two marijuana compounds of particular medical interest are delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD). A gas chromatography-tandem mass spectrometry (GC-MS-MS) method was developed to test for CBD, THC, hydroxy-THC (OH-THC) and carboxy-THC (COOH-THC) in human plasma. Calibrators (THC and OH-THC, 0.1 to 100; CBD, 0.25 to 100; COOH-THC, 0.5-500 ng/mL) and controls (0.3, 5 and 80 ng/mL, except COOH-THC at 1.5, 25 and 400 ng/mL) were prepared in blank matrix. Deuterated (d3) internal standards were added to 1-mL samples. Preparation involved acetonitrile precipitation, liquid-liquid extraction (hexane:ethyl acetate, 9:1), and MSTFA derivatization. An Agilent 7890 A GC was interfaced with an Agilent 7000 MS Triple Quadrupole. Selected reaction monitoring was employed. Blood samples were provided from a marijuana smoking study (two participants) and a CBD ingestion study (eight participants). Three analytes with the same transitions (THC, OH-THC and COOH-THC) were chromatographically separated. Matrix selectivity studies showed endogenous chromatographic peak area ratios (PAR) at the analyte retention times were <20% of the analyte limit of quantitation PAR. The intra-assay accuracy ranged from 83.5% to 118% of target and the intra-run imprecision ranged from 2.0% to 19.1%. The inter-assay accuracy ranged from 90.3% to 104% of target and the inter-run imprecision ranged from 6.5% to 12.0%. Stability was established for 25 hours at room temperature, 207 days at -20°C, after three freeze-thaw cycles and for 26 days for rederivatized processed samples. After smoking marijuana predictable concentrations of THC, OH-THC and COOH-THC were seen; low concentrations of CBD were detected at early time points. In moderate users who had not smoked for at least 9 hours before ingesting an 800 mg oral dose of CBD, the method was sensitive enough to follow residual concentrations of THC and OH-THC; sustained COOH-THC concentrations over 50 ng/mL validated its higher analytical range.

Cannabinoids assessment in plasma and urine by high performance liquid chromatography-tandem mass spectrometry after molecularly imprinted polymer microsolid-phase extraction.

A molecularly imprinted polymer (MIP) selective for cannabinoids [Δ9-tetrahydrocannabinol (Δ9-THC), 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (Δ9-THC-COOH), and 11-hydroxy-Δ9-tetrahydrocannabinol (Δ9-THC-OH)] has been synthesized, fully characterized, and applied to the assessment of plasma and urine analysis of marijuana abuse by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Δ9-THC-COOH was used as a template molecule, whereas ethylene glycol dimethacrylate (EGDMA) was used as a functional monomer, divinylbenzene (DVB) as a cross-linker, and 2,2'-azobisisobutyronitrile (AIBN) as an initiator. The prepared MIP was found to be highly selective for cannabinoids typically found in blood and urine, and also for cannabinol (CBN) and cannabidiol (CBD). MIP beads (50 mg) were loaded inside a cone-shaped device made of a polypropylene (PP) membrane for microsolid-phase extraction (µ-SPE) in batch mode. Optimum retention of analytes (0.1 to 1.0 mL of plasma/urine) was achieved by fixing plasma/urine pH at 6.5 and assisting the procedure by mechanical shaking (150 rpm, 40 °C, 12 min). Optimum elution conditions implied 2 mL of a 90:10 methanol/acetic acid and ultrasound extraction (35 kHz, 325 W) for 6 min. Good precision was assessed by intra-day and inter-day assays. In addition, the method was found to be accurate after intra-day and inter-day analytical recovery assays and after analyzing control serum and urine control samples. The limits of quantification were in the range of 0.36-0.49 ng L-1 (plasma analysis) and 0.47-0.57 ng L-1 (urine analysis). These values are low enough for confirmative conclusions regarding marijuana abuse through blood and urine analysis. Graphical Abstract ᅟ.

Free and Glucuronide Whole Blood Cannabinoids' Pharmacokinetics after Controlled Smoked, Vaporized, and Oral Cannabis Administration in Frequent and Occasional Cannabis Users: Identification of Recent Cannabis Intake.

BACKGROUND: There is increasing interest in markers of recent cannabis use because following frequent cannabis intake, Δ9-tetrahydrocannabinol (THC) may be detected in blood for up to 30 days. The minor cannabinoids cannabidiol, cannabinol (CBN), and THC-glucuronide were previously detected for ≤2.1 h in frequent and occasional smokers' blood after cannabis smoking. Cannabigerol (CBG), Δ9-tetrahydrocannabivarin (THCV), and 11-nor-9-carboxy-THCV might also be recent use markers, but their blood pharmacokinetics have not been investigated. Additionally, while smoking is the most common administration route, vaporization and edibles are frequently used. METHODS: We characterized blood pharmacokinetics of THC, its phase I and phase II glucuronide metabolites, and minor cannabinoids in occasional and frequent cannabis smokers for 54 (occasional) and 72 (frequent) hours after controlled smoked, vaporized, and oral cannabis administration. RESULTS: Few differences were observed between smoked and vaporized blood cannabinoid pharmacokinetics, while significantly greater 11-nor-9-carboxy-THC (THCCOOH) and THCCOOH-glucuronide concentrations occurred following oral cannabis. CBG and CBN were frequently identified after inhalation routes with short detection windows, but not detected following oral dosing. Implementation of a combined THC ≥5 µg/L plus THCCOOH/11-hydroxy-THC ratio <20 cutoff produced detection windows <8 h after all routes for frequent smokers; no occasional smoker was positive 1.5 h or 12 h following inhaled or oral cannabis, respectively. CONCLUSIONS: Vaporization and smoking provide comparable cannabinoid delivery. CBG and CBN are recent-use cannabis markers after cannabis inhalation, but their absence does not exclude recent use. Multiple, complimentary criteria should be implemented in conjunction with impairment observations to improve interpretation of cannabinoid tests. Clinicaltrials.gov Identifier: NCT02177513.