Pharmacology and toxicology of xylazine: quid novum?

The current opioid overdose crisis is characterized by the presence of unknown psychoactive adulterants. Xylazine is an alpha-2 receptor agonist that is not approved for human use but is commonly used in veterinary medicine due to its sedative and muscle-relaxant properties. Cases of human intoxication due to accidental or voluntary use have been reported since the 1980s. However, reports of adulteration of illicit opioids (heroin and illicit fentanyl) with xylazine have been increasing all over Western countries. In humans, xylazine causes respiratory depression, bradycardia, and hypotension-posing individuals, using xylazine-adulterated opioids. We present a narrative review of the latest intoxication cases related to xylazine, to bring awareness to readers and also to help pathologists to detect and deal with xylazine cases

Stability and Degradation Pathways of Different Psychoactive Drugs in Neat and in Buffered Oral Fluid

Sampling and drug stability in oral fluid (OF) are crucial factors when interpreting forensic toxicological analysis, mainly because samples may not be analyzed immediately after collection, potentially altering drug concentrations. Therefore, the stability of some common drugs of abuse (morphine, codeine, 6-monoacetylmorphine, cocaine, benzoylecgonine, Δ9-tetrahydrocannabinol, cannabidiol, amphetamine, 3,4-methylenedioxymethamphetamine, ketamine) and the more commonly consumed new psychoactive substances in our environment (mephedrone, and N-(adamantan-1-yl)-1-(5-fluoropentyl)-1H-indazole-3-carboxamide 5F-AKB48 also known as 5F-APINACA) was investigated in an OF pool for the presence and absence of M3 Reagent Buffer® up to 1 year of storage. Fortified OF samples were stored at three different temperatures (room temperature, 4 and -20°C) to determine the best storage conditions over time. Control fortified OF samples were stored at -80°C for reference purposes. Compounds with concentration changes within ±15% of initial value were considered stable. The drugs were significantly more stable in M3 Reagent Buffer® than in neat OF samples in all storage conditions. All analytes were stable for 1 year at 4°C and -20°C in M3 Reagent Buffer®. Drugs stability in OF varied depending on the analyte, the presence of a stabilizer, the storage duration and temperature. When immediate sample analysis is not possible, we suggest to store OF samples at 4 or -20°C and test them within 2 weeks. Alternatively, OF samples may be stored at 4 or -20°C with M3 Reagent Buffer® to be tested within 1 year

Dilute and shoot ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC–MS/MS) analysis of psychoactive drugs in oral fluid

A fast and sensitive ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC–MS/MS) method was developed and validated for the measurement of the most common drugs of abuse and some new psychoactive substances in oral fluid. The target compounds were 6-monoacetylmorphine, morphine, codeine, cocaine, benzoylecgonine, amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine, Δ-9-tetrahydrocannabinol, cannabidiol, mephedrone, ketamine and synthetic cannabinoid 5F-AKB48 (5F-APINACA). Oral fluid (OF) samples were 1/3 diluted with water and separated by reversed phase chromatography with gradient elution of 0.1% formic acid in water and 0.1% formic acid in acetonitrile and detected with tandem mass spectrometry operated in positive multiple reaction monitoring mode. The method was linear for all analytes under investigation from limit of quantification (LOQ, range: 0.5–5 ng/ml) to 250 ng/mL OF. Recovery of analytes under investigation and matrix effect were always higher than 90% (recovery range: 90.6–105.5% and matrix effect range 90.6–101.3%) whereas intra-assay and inter-assay precision and accuracy were always better than 15%. The developed method was successfully applied to ten OF specimens obtained from a proficiency test program and previously analyzed by gas-chromatography mass spectrometry. Analytes concentration between the two methods presented an excellent agreement (r 2 = 0.952) with overlapping values demonstrating method feasibility for high throughput laboratories needing indisputable results for clinical and/or forensic purposes. © 2019 Elsevier B.V

UHPLC-MS/MS analysis of cannabidiol and its metabolites in serum of patients with resistant epilepsy treated with CBD formulations

none13noCannabidiol (CBD) is a promising therapeutic agent with analgesic, myorelaxant, and anti-epileptic actions. Recently, a purified form of CBD (Epidiolex®) has been approved by the Eu-ropean Medicines Agency (EMA) for the treatment of two highly-refractory childhood-onset epi-lepsies (Dravet and Lennox-Gastaut syndrome). Given the interindividual response and the rela-tionship between the dose administered and CBD blood levels, therapeutic drug monitoring (TDM) is a valuable support in the clinical management of patients. We herein report for the first time a newly developed and validated method using ultra-high-performance liquid chromatography cou-pled with tandem mass spectrometry (UHPLC–MS/MS) to evaluate CBD and its metabolites (i.e., cannabidiol-7-oic acid (7-COOH-CBD), 7-hydroxycannabidiol (7-OH-CBD), 6-α-hydroxycanna-bidiol (6-α–OH–CBD) and 6-β-hydroxycannabidiol (6-β–OH–CBD)) in serum samples. The method reached the sensitivity needed to detect minimal amounts of analytes under investigation with lim-its of quantification ranging from 0.5 to 20 ng/mL. The validation results indicated in this method were accurate (average inter/intra-day error, <15%), precise (inter/intra-day imprecision, <15%), and fast (8 min run time). The method resulted to be linear in the range of 1–10,000 ng/mL for CBD-COOH, 1–500 ng/mL for 7-OH-CBD and CBD and 1–25 ng/mL for 6-α–OH–CBD and 6-β–OH–CBD. Serum levels of CBD (88.20–396.31 and 13.19–170.63 ng/mL) as well as of 7-OH-CBD (27.11–313.63 and 14.01–77.52 ng/mL) and 7-COOH-CBD (380.32–10,112.23 and 300.57–2851.82 ng/mL) were sig-nificantly higher (p < 0.05) in patients treated with GW pharma CBD compared to those of patients treated with galenic preparations. 6-α–OH–CBD and 6-β–OH–CBD were detected in the first group and were undetectable in the second group. 7-COOH-CBD was confirmed as the most abundant metabolite in serum (5–10 fold higher than CBD) followed by 7-OH-CBD. A significant correlation (p < 0.05) between the dose administrated and a higher bioavailability was confirmed in patients treated with a GW pharma CBD preparation.noneMalaca S.; Gottardi M.; Pigliasco F.; Barco S.; Cafaro A.; Amadori E.; Riva A.; Marcenaro M.; Striano P.; Cangemi G.; Pacifici R.; Pichini S.; Busardo F.P.Malaca, S.; Gottardi, M.; Pigliasco, F.; Barco, S.; Cafaro, A.; Amadori, E.; Riva, A.; Marcenaro, M.; Striano, P.; Cangemi, G.; Pacifici, R.; Pichini, S.; Busardo, F. P

UHPLC-MS/MS analysis of cannabidiol metabolites in serum and urine samples. Application to an individual treated with medical cannabis

No analytical assay is currently available for the simultaneous determination of CBD major metabolites in serum or urine samples of individuals treated with medical cannabis or CBD-based pharmaceuticals. We developed and validated a method using ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC–MS/MS) for quantifying cannabidiol (CBD) and its metabolites, cannabidiol-7-oic acid (7-COOH-CBD), 7- hydroxycannabidiol (7-OH-CBD), 6-alpha-hydroxycannabidiol (6-α–OH–CBD) and 6-beta-hydroxycannabidiol (6-β–OH–CBD) in serum and urine samples of an individual treated with medical cannabis. The ionization was performed by electrospray in negative mode to reach the sensitivity required to detect trace amounts, with limits of quantification ranging from 0.05 to 0.1 ng/mL. The method is accurate (average inter/intra-day error, <15%), precise (inter/intra-day imprecision, <15%) and fast (8 min run time) and it is an essential tool to investigate CBD pharmacokinetics and pharmacodynamics in individuals treated with medical cannabis or with CBD-based medical preparations

Cannabidiol, ∆9-tetrahydrocannabinol, and metabolites in human blood by volumetric absorptive microsampling and LC-MS/MS following controlled administration in epilepsy patients

: Cannabidiol (CBD) exhibits anti-inflammatory, anxiolytic, antiseizure, and neuroprotective proprieties without addictive or psychotropic side effects, as opposed to Δ9-tetrahydrocannabinol (THC). While recreational cannabis contains higher THC and lower CBD concentrations, medical cannabis contains THC and CBD in different ratios, along with minor phytocannabinoids, terpenes, flavonoids and other chemicals. A volumetric absorptive microsampling (VAMS) method combined with ultra-high-performance liquid chromatography coupled with mass spectrometry in tandem for quantification of CBD, THC and their respective metabolites: cannabidiol-7-oic acid (7-COOH-CBD); 7-hydroxy-cannabidiol (7-OH-CBD); 6-alpha-hydroxy-cannabidiol (6-α-OH-CBD); and 6-beta-hydroxycannabidiol (6-β-OH-CBD); 11- Hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC) and 11-Nor-9-carboxy-Δ9-tetrahydrocannabinol (THCCOOH). After overnight enzymatic glucuronide hydrolysis at 37°C, samples underwent acidic along with basic liquid-liquid extraction with hexane: ethyl acetate (9:1, v/v). Chromatographic separation was carried out on a C18 column, with the mass spectrometer operated in multiple reaction monitoring mode and negative electrospray ionization. Seven patients with intractable epilepsy were dosed with various CBD-containing formulations and blood collected just before their daily morning administration. The method was validated following international guidelines in toxicology. Linear ranges were (ng/ml) 0.5-25 THC, 11-OH-THC, THCCOOH, 6-α-OH-CBD and 6-β-OH-CBD; 10-500 CBD and 7-OH-CBD; and 20-5000 7-COOH-CBD. 7-COOH-CBD was present in the highest concentrations, followed by 7-OH-CBD and CBD. This analytical method is useful for investigating CBD, THC and their major metabolites in epilepsy patients treated with CBD preparations employing a minimally invasive microsampling technique requiring only 30 µL blood