Disposition of Hexahydrocannabinol Epimers and Their Metabolites in Biological Matrices following a Single Administration of Smoked Hexahydrocannabinol: A Preliminary Study.

In 2023, hexahydrocannabinol (HHC) attracted the attention of international agencies due to its rapid spread in the illegal market. Although it was discovered in 1940, less is known about the pharmacology of its two naturally occurring epimers, 9(R)-HHC and 9(S)-HHC. Thus, we aimed to investigate the disposition of hexahydrocannabinol epimers and their metabolites in whole blood, urine and oral fluid following a single controlled administration of a 50:50 mixture of 9(R)-HHC and 9(S)-HHC smoked with tobacco. To this end, six non-user volunteers smoked 25 mg of the HHC mixture in 500 mg of tobacco. Blood and oral fluid were sampled at different time points up to 3 h after the intake, while urine was collected between 0 and 2 h and between 2 and 6 h. The samples were analyzed with a validated HPLC-MS/MS method to quantify 9(R)-HHC, 9(S)-HHC and eight metabolites. 9(R)-HHC showed the highest Cmax and AUC0-3h in all the investigated matrices, with an average concentration 3-fold higher than that of 9(S)-HHC. In oral fluid, no metabolites were detected, while they were observed as glucuronides in urine and blood, but with different profiles. Indeed, 11nor-9(R)-HHC was the most abundant metabolite in blood, while 8(R)OH-9(R) HHC was the most prevalent in urine. Interestingly, 11nor 9(S) COOH HHC was detected only in blood, whereas 8(S)OH-9(S) HHC was detected only in urine.

Diagnostic Methods in Forensic Pathology: Autoptic Findings and Immunohistochemical Study in Cases of Sudden Death Due to a Colloid Cyst of the Third Ventricle.

The colloid cyst is a non-malignant tumor growth made of a gelatinous material covered by a membrane of epithelial tissue. It is usually located posterior to the foramen of Monro, in the anterior aspect of the third ventricle of the brain. Due to its location, it can cause obstructive hydrocephalus, increased intracranial pressure, and sudden cardiac death, catecholamine-mediated, through hypothalamus compression. All the mechanisms are still controversial, but the role of catecholamine has been confirmed with histological findings that highlighted myocardial injury (coagulative myocytolysis and contraction band necrosis, CBN). This study presents a case of sudden death in a previously healthy 22-year-old male due to a colloid cyst of the third ventricle. A complete autopsy was performed, highlighting in the brain an abundant quantity of cerebrospinal fluid (CSF) and a 2 cm pale grayish-green rounded cyst formation partially filling and distending the third ventricle. The diagnosis was confirmed through immunohistochemical investigation: positivity for Periodic acid-Schiff (PAS) staining and CK7 expression. In cases such as the one reported here, a combined approach of autopsy, histology, and immunohistochemistry is mandatory in order to identify the neoformation's location and morpho-structural characteristics for a correct differential diagnosis, as well as to identify the cause of death.

The first LC-MS/MS stereoselective bioanalytical methods to quantitatively detect 9R- and 9S-hexahydrocannabinols and their metabolites in human blood, oral fluid and urine.

A sensitive LC-MS/MS method for the simultaneous quantification of the (9 R)- and (9 S)- hexahydrocannabinols (HHCs), and their metabolites, in human urine, oral fluid (OF) and blood samples were developed, validated and used to the biological samples of volunteers. The analytes were extracted from 100 µL human samples. An isocratic elution mode with methanol was used for chromatographic separation of (9 R)- and (9 S)-HHC on an immobilized amylose tris(3-chloro-5-methylphenylcarbamate)-based chiral column Lux i-Amylose-3. The flow-rate of the mobile phase was 0.5 mL/min. An isocratic elution mode of methanol and water (80/20, v/v) was used for chromatographic separation of metabolites of (9 R)- and (9 S)-HHC on a Lux AMP chiral column (with a proprietary chiral selector) at a flow rate of 0.5 mL/min. MS/MS analysis was performed in positive ionization mode for HHC epimers, while in negative ionization mode was used for metabolites of HHCs. The calibration curves for HHCs and their metabolites in human samples ranged from 0.25- 240 ng mL-1 and 1 - 100 ng mL-1, respectively, with determination coefficients (r2) of ≥ 0.99. All analytes were stable at room temperature, 4 °C, in the autosampler (+10 °C) and -20 °C for 24 h, after three freeze/thaw cycles, and when stored at -20 °C up to one week after quality control (QC) sample preparation (concentration differences less than 20% with respect to time zero response), in blood, urine and OF.

Synthetic Cathinones and Neurotoxicity Risks: A Systematic Review.

According to the EU Early Warning System (EWS), synthetic cathinones (SCs) are the second largest new psychoactive substances (NPS) class, with 162 synthetic cathinones monitored by the EU EWS. They have a similar structure to cathinone, principally found in Catha Edulis; they have a phenethylamine related structure but also exhibit amphetamine-like stimulant effects. Illegal laboratories regularly develop new substances and place them on the market. For this reason, during the last decade this class of substances has presented a great challenge for public health and forensic toxicologists. Acting on different systems and with various mechanisms of action, the spectrum of side effects caused by the intake of these drugs of abuse is very broad. To date, most studies have focused on the substances' cardiac effects, and very few on their associated neurotoxicity. Specifically, synthetic cathinones appear to be involved in different neurological events, including increased alertness, mild agitation, severe psychosis, hyperthermia and death. A systematic literature search in PubMed and Scopus databases according to PRISMA guidelines was performed. A total of 515 studies published from 2005 to 2022 (350 articles from PubMed and 165 from Scopus) were initially screened for eligibility. The papers excluded, according to the criteria described in the Method Section (n = 401) and after full text analyses (n = 82), were 483 in total. The remaining 76 were included in the present review, as they met fully the inclusion criteria. The present work provides a comprehensive review on neurotoxic mechanisms of synthetic cathinones highlighting intoxication cases and fatalities in humans, as well as the toxic effects on animals (in particular rats, mice and zebrafish larvae). The reviewed studies showed brain-related adverse effects, including encephalopathy, coma and convulsions, and sympathomimetic and hallucinogenic toxidromes, together with the risk of developing excited/agitated delirium syndrome and serotonin syndrome.

Quantification of Carbonic Anhydrase Inhibitors and Metabolites in Urine and Hair of Patients and Their Relatives.

Carbonic anhydrase inhibitors (CAIs) are prescription drugs also used in doping to dilute urine samples and tamper with urinalyses. Dorzolamide, brinzolamide, and acetazolamide are prohibited by the World Anti-Doping Agency. Detecting CAIs and their metabolites in biological samples is crucial to documenting misuse in doping. We quantified dorzolamide, brinzolamide, acetazolamide, and their metabolites in the urine and hair of 88 patients under treatment for ocular hypertension or glaucoma. Samples of the patients' relatives were analyzed to assess potential for accidental exposure. After washing, 25 mg hair was incubated with an acidic buffer at 100 °C for 1 h. After cooling and centrifugation, the supernatant was analyzed by ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Urine (100 µL) was diluted and centrifuged before UHPLC-MS/MS analysis. Run time was 8 min through a reverse-phase column with a mobile phase gradient. MS/MS analysis was performed in a multiple-reaction monitoring mode after positive electrospray ionization. Median urinary concentration was 245 ng/mL (IQR: 116.2-501 ng/mL) for dorzolamide, 81.1 ng/mL (IQR: 35.9-125.3 ng/mL) for N-deethyl-dorzolamide, 0.77 ng/mL (IQR: 0.64 ng/mL-0.84 ng/mL) for N-acetyl-dorzolamide, 38.9 ng/mL (IQR: 20.4-79.2 ng/mL) for brinzolamide, and 72.8 ng/mL (IQR: 20.7-437.3 ng/mL) for acetazolamide. Median hair concentration was 0.48 ng/mg (IQR: 0.1-0.98 ng/mg) for dorzolamide, 0.07 ng/mg (IQR: 0.06-0.08 ng/mg) for N-deethyl-dorzolamide, 0.40 ng/mL (IQR: 0.13-1.95 ng/mL) for brinzolamide. Acetazolamide was detected in only one hair sample. Dorzolamide and brinzolamide were detected in the urine of three and one relatives, respectively. Cutoff concentrations of urinary dorzolamide and brinzolamide are necessary to preclude false positives due to contamination or passive exposure. We reported the first concentrations of brinzolamide in hair.