Development and validation of an HPLC-DAD method for the quantification of cannabigerol, cannabidiol, cannabinol and cannabichromene in human plasma and mouse matrices.

Cannabigerol, cannabidiol, cannabinol and cannabichromene are non-psychoactive phytocannabinoids, highly present in Cannabis sativa, for which numerous therapeutical applications have been described. However, additional pre-clinical and clinical data, including toxicopharmacokinetic and pharmacodynamic studies, remain required to support their use in clinical practice and new therapeutic applications. To support these studies, a new high performance liquid chromatography technique (HPLC) with diode-array detection (DAD) was developed and validated to quantify these cannabinoids in human plasma and mouse matrices. Sample extraction was accomplished by protein precipitation and double liquid-liquid extraction. Simvastatin and perampanel were used as internal standards in human and mouse matrices, respectively. Chromatographic separation was achieved in 16 min on an InfinityLab Poroshell® 120 C18 column (4.6 mm × 100 mm, 2.7 µm) at 40 °C. A mobile phase composed of water/acetonitrile was pumped with a gradient elution program at 1.0 mL min-1. The technique revealed linearity in the defined concentration ranges with a determination coefficient of over 0.99. Intra and inter-day accuracy and precision values ranged from -14.83 to 13.97% and 1.08 to 13.74%, respectively. Sample stability was assessed to ensure that handling and storage conditions did not compromise analyte concentrations in different matrices. Carry-over was absent and recoveries were over 77.31%. This technique was successfully applied for the therapeutic monitoring of cannabidiol and preliminary pre-clinical studies with cannabigerol and cannabidiol. All samples were within calibration ranges, with the exception of cannabigerol after intraperitoneal administration. This is the first HPLC-DAD technique that simultaneously quantifies cannabinoids in these biological matrices, supporting future pre-clinical and clinical investigations.

LC-MS-MS quantification of Δ8-THC, Δ9-THC, THCV isomers and their main metabolites in human plasma.

In recent years, potential therapeutic applications of several different cannabinoids, such as Δ9-tetrahydrocannabinol (Δ9-THC), its isomer Δ8-THC and Δ9-tetrahydrocannabivarin (Δ9-THCV), have been investigated. Nevertheless, to establish dose-effect relationship and to gain knowledge of their pharmacokinetics and metabolism, sensitive and specific analytical assays are needed to measure these compounds in patients. For this reason, we developed and validated an online extraction high-performance liquid/liquid chromatography-tandem mass spectrometry (LC/LC-MS-MS) method for the simultaneous quantification of 13 cannabinoids and metabolites including the Δ8 and Δ9 isomers of THC, THCV and those of their major metabolites in human plasma. Plasma was fortified with cannabinoids at varying concentrations within the working range of the respective compound and 200 µL was extracted using a simple one-step protein precipitation procedure. The extracts were analyzed using online trapping LC/LC-atmospheric pressure chemical ionization-MS-MS running in the positive multiple reaction monitoring mode. The lower limit of quantification ranged from 0.5 to 2.5 ng/mL, and the upper limit of quantification was 400 ng/mL for all analytes. Inter-day analytical accuracy and imprecision ranged from 82.9% to 109% and 4.3% to 20.3% (coefficient of variance), respectively. Of 534 plasma samples following controlled oral administration of Δ8-THCV, 236 were positive for Δ8-THCV (median; interquartile ranges: 3.5 ng/mL; 1.8-11.9 ng/mL), 383 for the major metabolite (-)-11-nor-9-carboxy-Δ8-tetrahydrocannabivarin (Δ8-THCV-COOH) (95.4 ng/mL; 20.7-328 ng/mL), 260 for (-)-11-nor-9-carboxy-Δ9-tetrahydrocannabivarin (Δ9-THCV-COOH) (5.8 ng/mL; 2.5-16.1 ng/mL), 157 for (-)-11-hydroxy-Δ8-tetrahydrocannabivarin (11-OH-Δ8-THCV) (1.7 ng/mL; 1.0-3.7 ng/mL), 49 for Δ8-THC-COOH (1.7 ng/mL; 1.4-2.3 ng/mL) and 42 for Δ9-THCV (1.3 ng/mL; 0.8-1.6 ng/mL). We developed and validated the first LC/LC-MS-MS assay for the specific quantification of Δ8-THC, Δ9-THC and THCV isomers and their respective metabolites in human plasma. Δ8-THCV-COOH, 11-hydroxy-Δ8-THCV and Δ9-THCV-COOH were the major Δ8-THCV metabolites in human plasma after oral administration of 98.6% pure Δ8-THCV.

Cross-reactivity of 24 cannabinoids and metabolites in blood using the Immunalysis Cannabinoids Direct enzyme-linked immunosorbent assay.

With wider availability of synthetic and semi-synthetic cannabinoids in the consumer space, there is a growing impact on public health and safety. Forensic toxicology laboratories should keep these compounds in mind as they attempt to remain effective in screening for potential sources of human performance impairment. Enzyme-linked immunosorbent assay (ELISA) is a commonly utilized tool in forensic toxicology, as its efficiency and sensitivity make it useful for rapid and easy screening for a large number of drugs. This screening technique has lower specificity, which allows for broad cross-reactivity among structurally similar compounds. In this study, the Cannabinoids Direct ELISA kit from Immunalysis was utilized to assess the cross-reactivities of 24 cannabinoids and metabolites in whole blood. The assay was calibrated with 5 ng/mL of 11-nor-9-carboxy-Δ9-tetrahydrocannabinol and the analytes of interest were evaluated at concentrations ranging from 5 to 500 ng/mL. Most parent compounds demonstrated cross-reactivity ≥20 ng/mL, with increasing alkyl side-chain length relative to Δ9-tetrahydrocannabinol resulting in decreased cross-reactivity. Of the 24 analytes, only the carboxylic acid metabolites, 11-nor-9-carboxy-Δ8-tetrahydrocannabinol, 11-nor-9(R)-carboxy-hexahydrocannabinol and 11-nor-9(S)-carboxy-hexahydrocannabinol, were cross-reactive at levels ≤10 ng/mL. Interestingly, 11-nor-9(R)-carboxy-hexahydrocannabinol demonstrated cross-reactivity at 5 ng/mL, where its stereoisomer 11-nor-9(S)-carboxy-hexahydrocannabinol, did not. As more information emerges about the prevalence of these analytes in blood specimens, it is important to understand and characterize their impact on current testing paradigms.

Insights into the human metabolism of hexahydrocannabinol by non-targeted liquid chromatography-high-resolution tandem mass spectrometry.

Hexahydrocannabinol (HHC), 6,6,9-trimethyl-3-pentyl-6a,7,8,9,10,10a-hexahydrobenzo[c]chromen-1-ol, is a semi-synthetic cannabinoid that has presented challenges to analytical laboratories due to its emergence and spread in the drug market. The lack of information on human pharmacokinetics hinders the development and application of presumptive and confirmatory tests for reliably detecting HHC consumption. To address this knowledge gap, we report the analytical results obtained from systematic forensic toxicological analysis of body-fluid samples collected from three individuals suspected of drug-impaired driving after HHC consumption. Urine and plasma samples were analyzed using non-targeted liquid chromatography-high-resolution tandem mass spectrometry. The results provided evidence that HHC undergoes biotransformation reactions similar to other well-characterized cannabinoids, such as ∆9-tetrahydrocannabinol or cannabidiol. Notably, HHC itself was only detectable in plasma samples, not in urine samples. The observed Phase I reactions involved oxidation of C11 and the pentyl side chain, leading to corresponding hydroxylated and carboxylic acid species. Additionally, extensive glucuronidation of HHC and its Phase I metabolites was evident.

GC-MS/MS analysis of synthetic cannabinoids 5F-MDMB-PICA and 5F-CUMYL-PICA in forensic cases.

Aim: Validate a method to quantify 1-(5-fluoropentyl)-N-(2-phenylpropan-2-yl)-1H-indole-3-carboxamide (5F-CUMYL-PICA) and methyl 2-[[1-(5-fluoropentyl) indole-3-carbonyl] amino]-3,3-dimethyl-butanoate (5F-MDMB-PICA) in blood samples using GC-MS/MS. Materials & methods: A solid-phase extraction (SPE) method has been developed to quantify 5F-MDMB-PICA and 5F-CUMYL-PICA in authentic human blood samples. Results & conclusion: The limit of detection (LOD) was 0.1 and 0.11 ng/ml for 5F-CUMYL-PICA and 5F-MDMB-PICA, respectively, while the limit of quantification (LOQ) was 0.50 ng/ml for both two compounds. Recovery was 91.40, 82.54 and 85.10% for SPE, supported liquid extraction (SLE) and ISOLUTE C18; matrix effects 15, 24 and 22.5% for SPE, SLE and ISOLUTE C18; accuracy was 2.4-5.5 and 3.9-7.3% for SPE, SLE and ISOLUTE C18, while precision was 4.6-7.7 and 6.4-8.3% for SPE, SLE and ISOLUTE C18, respectively. The concentrations of 5F-CUMYL-PICA and 5F-MDMB-PICA in the authentic human blood samples were 2.18 and 3.07 ng/ml, respectively. The validated method was successfully used in supporting the quantification of analytes in blood.

[Box: see text].

Pharmacokinetics and pharmacodynamics of the synthetic cannabinoid, 5F-MDMB-PICA, in male rats.

5F-MDMB-PICA is a popular synthetic cannabinoid associated with analytically confirmed intoxications. In vitro studies show 5F-MDMB-PICA is a potent cannabinoid-1 receptor (CB1) agonist, but little information is available about in vivo pharmacokinetics and pharmacodynamics. To this end, the present study had three aims: 1) to develop a validated method for detection of 5F-MDMB-PICA and its metabolites in rat plasma, 2) to utilize the method for investigating pharmacokinetics of 5F-MDMB-PICA in rats, and 3) to relate 5F-MDMB-PICA pharmacokinetics to pharmacodynamic effects. 5F-MDMB-PICA and its metabolites were quantified using liquid chromatography tandem mass spectrometry (LC-MS/MS) and method validation followed forensic standards. Male Sprague-Dawley rats bearing surgically implanted jugular catheters and subcutaneous (s.c.) temperature transponders received 5F-MDMB-PICA (50, 100, or 200 µg/kg, s.c.) or its vehicle. Blood samples were drawn at 15, 30, 60, 120, 240, and 480 min post-injection, and plasma was assayed using LC-MS/MS. At each blood draw, body temperature, and catalepsy scores were recorded. Maximum plasma concentrations (Cmax) of 5F-MDMB-PICA rose linearly with increasing dose (1.72-6.20 ng/mL), and plasma half-life (t1/2) ranged from 400 to 1000 min 5F-MDMB-PICA-3,3-dimethylbutanoic acid and 5OH-MDMB-PICA were the only metabolites detected, and plasma concentrations were much lower than the parent drug. 5F-MDMB-PICA induced robust hypothermia and catalepsy-like symptoms that were significantly correlated with concentrations of 5F-MDMB-PICA. Radioligand binding in rat brain membranes revealed 5F-MDMB-PICA displays high affinity for CB1 (IC50 = 2 nM) while metabolites do not. In summary, 5F-MDMB-PICA is a potent CB1 agonist in rats whose pharmacodynamic effects are related to circulating concentrations of the parent drug and not its metabolites.

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.

Detectability of cannabinoids in the serum samples of cannabis users: Indicators of recent cannabis use? A follow-up study.

Forensic toxicologists are frequently required to predict the time of last cannabis consumption. Several studies suggested the utility of minor cannabinoids as indicators of recent cannabis use. Because several factors influence blood cannabinoid concentrations, the interpretation of serum cannabinoid concentrations remains challenging. To assess the informative value of serum cannabinoid levels in cannabis users (in total N = 117 patients, including 56 patients who stated an exact time of last cannabis use within 24 h before blood sampling), the detectability of cannabinoids, namely, delta-9-tetrahydrocannabinol (delta-9-THC), 11-hydroxy-delta-9-THC, 11-nor-9-carboxy-delta-9-THC, cannabichromene (CBC), cannabidiol (CBD), cannabinol (CBN), cannabidivarin, tetrahydrocannabivarin, cannabigerol (CBG), cannabicyclol, delta-8-THC, tetrahydrocannabinolic acid A, cannabichromenic acid, cannabidiolic acid (CBDA), cannabigerolic acid, cannabicyclolic acid (CBLA), 11-nor-9-carboxy-THCV (THCVCOOH), and 11-nor-CBN-9-COOH, was investigated. Excluding CBDA and CBLA, all investigated cannabinoids were detected in at least one analyzed sample. The interval between cannabis consumption and sample collection (reported by the patients) was not correlated with cannabinoid concentrations. Minor cannabinoids tended to be more easily detected in samples obtained shortly after consumption. However, some samples tested positive for minor cannabinoids despite an interval of several hours or even days between consumption and sampling (according to patients' statements). For instance, CBC, CBG, THCVCOOH, CBD, and CBN in certain cases could be detected more than 24 h after the last consumption of cannabis. Thus, findings of minor cannabinoids should always be interpreted with caution.

Unintentional cannabis poisoning in toddlers: A one year study in Marseille.

France is the country with the highest prevalence of cannabis use in Europe, despite the fact that cannabis has not been legalized. This prevalence is still increasing along with THC content in cannabis products. In the meantime, unintentional cannabis poisoning by ingestion in toddlers is constantly rising. The aim of this study was to document children's cannabis poisoning biologically and clinically. Plasma and urine samples were extracted by solid phase extraction and analyzed by liquid chromatography coupled to tandem mass spectrometry. Children under 4 years old admitted in pediatric emergency departments for cannabis intoxication between February 1st 2019 and January 31st 2020 were included in this study. Twenty-six children were included (14 female and 12 male), the mean age was 17 months (10-41 months). THC, 11-OH-THC and THC-COOH plasma concentrations ranged from 2.9 to 93 ng/mL, 2.6-65 ng/mL and 29-914 ng/mL, respectively. The most frequent symptoms were drowsiness and hypotonia. Six critical cases were observed: 5 coma and 1 respiratory depression. All children having THC plasma concentrations over 60 ng/mL were in coma. Cannabis poisoning in toddlers become more frequent, 9 cases/year were reported in Marseille in 2007 and 26 cases/year in this study. There is a rising in severe clinical cases, particularly coma. These observations could be explained by an increase in THC content in cannabis products, and a trivialization of cannabis consumption. The unintentional ingestion of cannabis by children is a serious public health concern, and cannabis legalization could worsen this problem.

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.

Cannabis use and measurement of cannabinoids in plasma and breast milk of breastfeeding mothers.

BACKGROUND: Information on cannabinoids in breast milk and maternal cannabis use is limited. We quantified cannabinoids in plasma and breast milk of breastfeeding mothers and assessed cannabis use patterns. METHODS: This is a prospective study at a university hospital in a state with legal medical and recreational cannabis. Breast milk and plasma samples along with survey data were collected from volunteers using cannabis in the last 48 h at 2 weeks and 2 months postpartum. RESULTS: Twenty subjects were enrolled. Median age (IQR) was 27 (24-34) years. Median (IQR) instances of cannabis use in the last 7 days were visit 1: 17 (6-29) and visit 2: 23 (15-45). Median (IQR) tetrahydrocannabinol (THC) concentrations were: plasma 3.7 ng/ml (0.8-56.8) and breast milk 27.5 ng/ml (0.8-190.5). Median (IQR) cannabidiol (CBD) concentrations were: plasma 0.6 ng/ml (0.5-6.4) and breast milk 1.2 ng/ml (0.5-17.0). Median (IQR) THC M/P: 7.0 (1.8-34.6) and CBD M/P: 2.6. Median breast milk THC concentration increased from visit 1 to visit 2 by 30.2 ng/ml (95% CI 3.05-69.3 ng/ml). CONCLUSIONS: THC and CBD accumulate in breast milk. Breastfeeding mothers used cannabis frequently and increased use in the early postpartum period. Research on the effects of infant exposure to cannabinoids in breast milk is urgently needed. IMPACT: Cannabis use is increasing in the general population and many nursing mothers use cannabis. THC has been previously detected in breast milk but little is known on how it concentrates relative to plasma. Data on cannabinoids other than THC, reasons for cannabis use, and patterns of use in breastfeeding women are also limited. We detected THC and CBD in breast milk. Both concentrate in breast milk relative to plasma. We showed that breastfeeding mothers increased cannabis use in the weeks after childbirth. Further research is needed to evaluate infant exposure to cannabinoids via breast milk and effects on infant health.

Blood concentrations of synthetic cannabinoids.

INTRODUCTION: Synthetic cannabinoids (SCs) are the largest and most diverse group of new psychoactive substances. Their influence on organism is unpredictable and often lead to intoxications, including fatal poisonings. The interpretation of blood concentrations of detected SC although complicated, can help to determine the effects of an administered drug. The interpretation of one's own results usually requires a comparison to previously published cases, therefore, a referenced compilation of concentration ranges would be useful. MATERIAL AND METHODS: The data collection was based on a search of PubMed and Google search engine. All the available data from articles and reports where SCs concentrations have been measured in whole blood, serum or plasma were included in the data analysis. RESULTS: Presented table lists the observed concentrations in fatal and non-fatal cases involving 65 SCs. A reference list with original papers has been added for each compound, which makes it easy to find the source data. CONCLUSION: The observed concentrations of SCs vary widely and often have overlapping ranges for fatal and non-fatal cases. Conclusions regarding the cause of death are difficult based upon the concentrations alone and should include knowledge of the clinical situation in each case.

Evaluation of Synthetic Cannabinoid Metabolites in Human Blood in the Absence of Parent Compounds: A Stability Assessment.

Synthetic cannabinoids represent a chemically diverse class of novel psychoactive substances (NPS) responsible for large analytical and interpretative challenges for forensic toxicologists. Between 2016 and 2019, the three most prevalent synthetic cannabinoids in the United States were MMB-FUBINACA (FUB-AMB), 5F-MDMB-PINACA (5F-ADB) and 5F-MDMB-PICA, based on results from seized drug and toxicology testing. In 2018, accurate determination of synthetic cannabinoid positivity was brought into question as it was determined that the metabolites of these drug species were present in the absence of parent compounds in forensically relevant blood samples. During this study, the stability of MMB-FUBINACA, 5F-MDMB-PINACA and 5F-MDMB-PICA was evaluated, as well as the characterization of breakdown products. A liquid-liquid extraction method was assessed for recovery of basic parent compounds and acidic metabolites and deemed fit for use in this study. Analysis was performed by liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) using a SCIEX TripleTOF® 5600+. All three synthetic cannabinoids were found to be unstable when stored in blood at either room temperature or refrigerated; all analytes were considerably more stable when stored in the freezer. All three synthetic cannabinoids degraded to their respective butanoic acid metabolites: MMB-FUBINACA 3-methylbutanoic acid, 5F-MDMB-PINACA 3,3-dimethylbutanoic acid and 5F-MDMB-PICA 3,3-dimethylbutanoic acid. All three of these metabolites were studied and determined to be stable in blood at all storage conditions. Considering these results, our laboratory continued testing for synthetic cannabinoid metabolites in blood samples and found 83 positives (21%) for only a synthetic cannabinoid metabolite. A case report is presented herein where 5F-MDMB-PINACA 3,3-dimethylbutanoic acid was identified in the absence of 5F-MDMB-PINACA. Forensic toxicologists should be aware of the results of this study as they directly impact analytical consideration for test development and implementation, as well as interpretation of findings.

Differentiation of Positional Isomers of Halogenated Benzoylindole Synthetic Cannabinoid Derivatives in Serum by Hybrid Quadrupole/Orbitrap Mass Spectrometry.

Legally regulated synthetic cannabinoids (SCs) are continuously being created by making minor positional modifications to pre-existing analogs; thus, compounds with minor structural differences must be isolated and identified accurately. For iodo-benzoylindole derivatives of SCs, only specific isomers are currently the target of legal control, and it is necessary to establish an analytical method for accurately identifying positional isomers. In this study, we synthesized a series of 57 designer drugs and developed a screening method for identifying halogen positional isomers on the phenyl ring of benzoylindole derivative SCs in serum. Analytical methods using the Discovery F5 pentafluorophenyl column gave the best selectivity and retention of the positional isomer analytes. Some of the meta and para iodo-substituted SCs were eluted at similar retention times and were difficult to separate by liquid chromatography (LC). However, they were identified via the relative abundance of the two product ions in the collision-induced dissociation reaction using LC-hybrid quadrupole/orbitrap high-resolution mass spectrometry. Our synthesized halogen-substituted positional isomer SC library and method for differentiating positional isomers of halogenated benzoylindole SC derivatives could provide an indispensable analysis tool for identifying illegal drugs in serum of drug users.

Emergence of Cumyl-PEGACLONE-related fatalities in the Northern Territory of Australia.

Suspected unnatural or unexpected deaths in the Northern Territory of Australia are reportable to the coroner, and investigation of such cases typically includes a post-mortem examination with comprehensive toxicological screening. An autopsy case series of five Cumyl-PEGACLONE-related fatalities over a recent eighteen-month period is presented. Databases of the Northern Territory coroner's office and the Royal Darwin Hospital Forensic Pathology Unit were searched to identify deaths related to synthetic cannabis use between July 1, 2018 and December 31, 2020. Toxicological analysis was performed at Forensic Science South Australia using a combination of liquid chromatography, gas chromatography and mass spectrometry. Cumyl-PEGACLONE, a synthetic cannabinoid receptor agonist (SCRA) with a gamma-carbolinone core, was detected in five cases (range in post-mortem blood 0.73-3.0 µg/L). Concurrent alcohol use and underlying cardiovascular disease were considered relevant factors in most cases. Toxicological Significance Scoring was carefully considered in all five cases, and in four cases, the presence of Cumyl-PEGACLONE was considered to be highly significant (TSS = 3). Synthetic cannabis use has not previously been identified in Northern Territory drug trends, and only one fatality related to the use of gamma-carbolines was identified in a recent Australia-wide study on synthetic cannabinoid-related fatalities. Deaths related to Cumyl-PEGACLONE use are emerging in the Northern Territory of Australia; this has public health implications. Although the exact mechanism(s) of death related to Cumyl-PEGACLONE are not fully established, this additional descriptive case series reaffirm an association with underlying cardiovascular disease, and suggest that concurrent use with alcohol may be relevant.

Pupillary effects in habitual cannabis consumers quantified with pupillography.

Driving under the influence of alcohol (DUIA) and drugs (DUID) is considered an elevated risk for traffic safety. When assessing a driver's fitness to drive, standardized and objective measurement methods are still required, in order to clarify the question whether an individual is under the influence of substances acting on the central nervous system (CNS). We exposed healthy test subjects (n=41) as well as persons who were under the influence of cannabis after repeated inhalation to multiple light stimuli using infrared technology and measured the pupillary light reflex (PLR). Toxicological tests of blood samples taken from every subject followed. The aims of this study were to assess the differences in pupillography response between cannabis consumers after a washout period and no cannabis consumers as well as the dose related effects on pupillography parameters of cannabis in cannabis consumers. All four pupillary parameters changed according to a weakened pupil function after acute administration of cannabis in all test subjects. Furthermore, it could be observed that habitual cannabis consumers showed an altered pupillary function just before the first dose was taken, suggesting that the long-term effects and addiction also have to be taken into account, when effects of the CNS are discussed. The results of the present study show that almost all pupil parameters could be reliable indicators for the detection of subjects under the acute effect of cannabis.

GC-MS analysis of underivatised new psychoactive substances in whole blood and urine.

Herein a method was develop and validated for the detection and quantification of five new psychoactive substances (NPS) belonging to three categories: synthetic cathinones (mephedrone, 3,4-MDPV), opioids (AH-7921) and cannabinoids (JWH-018, AM-2201) by EI GC-MS. Target analytes were quantified in whole blood; in urine the same compounds plus methylone were detected. Liquid-liquid extraction by MTBE - butyl acetate (1:1, v/v) in blood and butyl acetate in urine was applied for the recovery of analytes, while no derivatization was necessary for their sensitive detection and quantification. The method showed good linearity for all analytes within a concentration range from 0.25 to 2 µg/mL for mephedrone, from 0.02 to 0.16 µg/mL for 3,4-MDPV and AH-7921 and from 0.005 to 0.04 µg/mL for JWH-018 and AM-2201. LOD ranged from 0.002 µg/mL (JWH-018 and AM-2201 in blood and urine), to 0.08 µg/mL (mephedrone in urine). LOQ in blood ranged from 0.005 µg/mL for JWH-018 and AM-2201 to 0.25 µg/mL for mephedrone. Accuracy was within acceptable limits with % bias ranging from +20% to -17.98% for intra-assay study and from +18.87% to -11.16% for inter-assay study. Precision was found to be between 2.60% and 17.17% (CV%) for intra-assay study and from 6.03% to 13.72% (CV%) for inter-assay study. An intra laboratory comparison provided proof of the method robustness. The developed method can be used for the reliable and fast quantification of five NPS in blood and the detection of six NPS in urine within the practice of a clinical or forensic toxicology laboratory.