Human metabolism of four synthetic benzimidazole opioids: isotonitazene, metonitazene, etodesnitazene, and metodesnitazene.

Following isotonitazene scheduling in 2019, the availability of alternative 2-benzylbenzimidazole opioids (nitazenes) on the global drug market increased, resulting in many fatalities worldwide. Nitazenes are potent µ-opioid receptor agonists with strong narcotic/analgesic effects, and their concentrations in biological matrices are low, making the detection of metabolite biomarkers of consumption crucial to document use in clinical and forensic settings. However, there is little to no data on the metabolism of the most recently available nitazenes, especially desnitro-analogues. The aim of the research was to assess isotonitazene, metonitazene, etodesnitazene, and metodesnitazene human metabolism and identify specific metabolite biomarkers of consumption. The four analogues were incubated with 10-donor-pooled human hepatocytes, and the incubates were analyzed by liquid chromatography-high-resolution tandem mass spectrometry and data mining with Compound Discoverer (Thermo Scientific); the analysis was supported by in silico metabolite predictions with GLORYx open-access software. Metabolites were identified in postmortem blood and/or urine samples from two metonitazene-positive and three etodesnitazene-positive cases following the same workflow, with and without glucuronide hydrolysis in urine, to confirm in vitro results. Twelve, nine, twenty-two, and ten metabolites were identified for isotonitazene, metonitazene, etodesnitazene, and metodesnitazene, respectively. The main transformations were N-deethylation at the N,N-diethylethanamine side chain, O-dealkylation, and further O-glucuronidation. In vitro and autopsy results were consistent, demonstrating the efficacy of the 10-donor-pooled human hepatocyte model to predict human metabolism. We suggest the parent and the corresponding O-dealkyl- and N-deethyl-O-dealkyl metabolites as biomarkers of exposure in urine after glucuronide hydrolysis, and the corresponding N-deethyl metabolite as additional biomarker in blood.

In silico, in vitro, and in vivo human metabolism of acetazolamide, a carbonic anhydrase inhibitor and common "diuretic and masking agent" in doping.

Acetazolamide (ACZ) is a carbonic anhydrase inhibitor prescribed for the treatment of various pathologies. It is also used in doping and is prohibited in and out of sportive competitions. ACZ was reported not to undergo metabolization. However, the detection of ACZ metabolites may be critical for documenting ACZ use. We aimed to further investigate ACZ metabolic fate in humans. ACZ putative metabolites were generated in silico to assist in metabolite identification. ACZ was incubated with primary human hepatocytes to identify in vitro metabolites (10 µmol/l ACZ and 106 cells/ml), and urine and plasma samples from patients receiving a single 5.0 mg/kg BW PO ACZ dose were analyzed to confirm the results in vivo. Analyses were performed with reversed-phase liquid chromatography and hydrophilic interaction chromatography coupled with high-resolution tandem mass spectrometry (RPLC-HRMS/MS and HILIC-HRMS/MS, respectively). Data were screened with a software-assisted targeted/untargeted workflow. ACZ was quantified in urine samples with creatinine normalization. We identified two metabolites in hepatocyte incubations and three additional metabolites in urine and plasma. Major transformations included cysteine conjugation, glucuronidation, and N-acetylation. All metabolites were detected in plasma, 1.5 h after intake. Major metabolites were detected in urine from 0.25 to 24 h (last collection) after intake. As opposed to the literature, ACZ does undergo metabolization in humans. We propose ACZ, ACZ-Cys, and N-acetyl-ACZ in urine, and ACZ and N-acetyl-ACZ in plasma as specific biomarkers of ACZ intake in doping.

A Review of Synthetic Cathinone-Related Fatalities From 2017 to 2020.

BACKGROUND: Synthetic cathinones (SCs) are designer analogs of the natural active principle of khat. Since their appearance on the black market in 2003, their popularity has increased annually, and they have become the most seized class of new psychoactive substances reported to the UNODC Early Warning Advisory system. The constant introduction of newly synthesized molecules makes this issue difficult to monitor. The authors reviewed the most recent SC-related fatalities worldwide to highlight new trends of consumption, reporting acute pharmacological and toxicological symptoms, scene investigations, analytical methods, and reported SC concentrations in diverse biological matrices. METHODS: A literature search was performed using scientific databases such as PubMed, Scopus, Science Direct, Web of Science, and Research Gate to identify relevant scientific publications from 2017 to 2020. In addition, a search was conducted through the EU EWS. RESULTS: From 2017 to 2020, 31 different SCs were identified in 75 reported fatal intoxications in the literature, alone or in combination with other substances. The most abused SCs were N-ethylpentylone, N-ethylhexedrone, and 4-chloromethcathinone. The EU EWS included less detail on 72 additional SC-related fatalities from 2017 to 2020. CONCLUSIONS: New SCs continuously replace older natural and synthetic stimulant drugs, making determining the cause of death difficult. Analytical methods and high-performance mass spectrometry instruments are essential to detect the low concentrations of these potent new SCs. Little data are available on the pharmacology of these new drugs; the evaluation of toxicological antemortem and postmortem findings provides critical data on the drug's pharmacology and toxicology and for the interpretation of new SC cases.

Disposition of cannabinoids and their metabolites in serum, oral fluid, sweat patch and urine from healthy individuals treated with pharmaceutical preparations of medical cannabis.

Recently, several countries authorized the use of cannabis flowering tops (dried inflorescences) with a standardized amount of Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD) and their acidic precursors [Δ-9-tetrahydrocannabinolic acid A (THCA-A) and cannabidiolic acid (CBDA)] to treat neurogenic pain. We studied the acute pharmacological effects and disposition of cannabinoids and their metabolites in serum, oral fluid, sweat patch and urine of 13 healthy individuals treated with medical cannabis decoction and oil. Cannabinoids and their metabolites were quantified by ultrahigh performance tandem mass spectrometry. Even if the oil contained a significantly higher amount of THC, the absorption of THC and its metabolites were similar in both herbal preparations. Conversely, whereas oil contained a significantly higher amount of CBD and a lower amount of CBDA, absorption was significantly higher after decoction intake. Only cannabinoids present in both herbal preparations (THC, CBD, THCA-A and CBDA) were found in oral fluid, due to the higher acidity compared with that of serum. THC metabolites urinary excretion was always higher after decoction administration. Decoction induced greater feeling of hunger and drowsiness than oil preparation. Pharmacokinetics of cannabinoids, their precursors and their metabolites in biological fluids of individuals treated with cannabis decoction and oil showed a high interindividual variability. The aqueous preparation was generally better absorbed than the oil, even if it contained a minor amount of THC, THCA-A and CBD.

Monitoring Perinatal Exposure to Cannabis and Synthetic Cannabinoids.

PURPOSE: Drug use during pregnancy is a critical global challenge, capable of severe impacts on neonatal development. However, the consumption of cannabis and synthetic cannabinoids is on the rise in pregnant women. Obstetric complications with increased risks of miscarriage, fetal growth restriction, and brain development impairment have been associated with perinatal cannabis exposure, but data on synthetic cannabinoid use during pregnancy are limited. METHODS: We reviewed studies that investigated the risks associated with cannabis and synthetic cannabinoid use and those that reported the concentrations of cannabinoids and synthetic cannabinoids in maternal (breast milk) and neonatal (placenta, umbilical cord, meconium, and hair) matrices during human pregnancy. A MEDLINE and EMBASE literature search to identify all relevant articles published in English from January 1998 to April 2019 was performed. RESULTS: Cannabis use during pregnancy is associated with increased risks of adverse obstetrical outcomes, although neurobehavioral effects are still unclear. Analyses of cannabinoids in meconium are well documented, but further research on other unconventional matrices is needed. Adverse effects due to perinatal synthetic cannabinoid exposure are still unknown, and analytical data are scarce. CONCLUSIONS: Awareness of the hazards of drug use during pregnancy should be improved to encourage health care providers to urge pregnant women to abstain from cannabis and, if cannabis-dependent, seek treatment. Moreover, substances used throughout pregnancy should be monitored as a deterrent to cannabis use, and potential cannabis-dependent women should be identified, so as to limit cannabis-fetal exposure during gestation, and provided appropriate treatment.

Δ9-Tetrahydrocannabinol and Cannabidiol Time Courses in the Sera of "Light Cannabis" Smokers: Discriminating Light Cannabis Use from Illegal and Medical Cannabis Use.

BACKGROUND: Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) time courses in serum and physiological and behavioral effects associated with smoking 1 or 4 "light cannabis" cigarettes were studied. Biomarkers to differentiate light cannabis versus illegal and medical cannabis use were also investigated. METHODS: Sera were obtained at different times from 6 healthy light cannabis consumers and 6 individuals who smoked 1 and 4 cigarettes, within 4 hours through a liquid-liquid method and analyzed by liquid chromatography-tandem mass spectrometry. RESULTS: In serum, minimal THC concentration was observed after a single cigarette smoke, while repeated smoking increased it by 1 order of magnitude. CBD concentrations were higher, but did not increase linearly, probably because it does not preferentially volatilize compared with THC. The highest THC and CBD concentrations were observed 0.5 hours after the start of the smoking of 1 cigarette. Serum THC ranged from 2.7 to 5.9 ng/mL, while serum CBD varied from 5.7 to 48.2 ng/mL. Similarly, the highest THC and CBD concentrations were observed 0.5 hours after the smoking of 4 cigarettes. Specifically, the ranges were THC: 11.0-21.8 ng/mL and CBD: 19.4-35.3 ng/mL. In both cases, the mean THC/CBD concentration ratio ranged from 0.2 to 0.9. There were no significant changes in blood pressure, heart rate, and body temperature, but participants who smoked 4 cigarettes experienced severe drowsiness. CONCLUSIONS: THC and CBD time courses in the sera of light cannabis smokers were similar to those previously observed in oral fluid and blood. Serum THC/CBD concentration ratio not higher than the mean value of 0.9 might be a useful biomarker to identify use of light cannabis versus that of illegal THC cannabis (where THC/CBD concentration ratios are generally greater than 10) or versus that of medical cannabis (where ratios are greater than 1). Consumers should be advised of possible drowsiness after he repeated smoking of light cannabis cigarettes.

Testing Unconventional Matrices to Monitor for Prenatal Exposure to Heroin, Cocaine, Amphetamines, Synthetic Cathinones, and Synthetic Opioids.

BACKGROUND: The prevalence of drug use during pregnancy continues to increase despite the associated serious adverse obstetrical outcomes, including increased risk of miscarriage, fetal growth restriction, brain development impairment, neonatal abstinence syndrome, preterm delivery, and stillbirths. Monitoring drug use during pregnancy is crucial to limit prenatal exposure and provide suitable obstetrical health care. The authors reviewed published literature reporting the concentrations of common drugs of abuse and new psychoactive substances (NPS), such as synthetic cathinones and synthetic opioids, NPS, and their metabolites using unconventional matrices to identify drug use during pregnancy and improve data interpretation. METHODS: A literature search was performed from 2010 to July 2019 using PubMed, Scopus, Web of Science scientific databases, and reports from international institutions to review recently published articles on heroin, cocaine, amphetamine, methamphetamine, synthetic cathinone, and synthetic opioid monitoring during pregnancy. RESULTS: Meconium has been tested for decades to document prenatal exposure to drugs, but data regarding drug concentrations in amniotic fluid, the placenta, the umbilical cord, and neonatal hair are still lacking. Data on prenatal exposure to NPS are limited. CONCLUSIONS: Maternal hair testing is the most sensitive alternative matrix for identifying drug use during pregnancy, while drug concentrations in the meconium, placenta, and umbilical cord offer the identification of prenatal drug exposure at birth. Adverse developmental outcomes for the infant make it critical to promptly identify maternal drug use to limit fetal exposure or, if determined at birth, to provide resources to the exposed child and family. Alternative matrices offer choices for monitoring and challenge laboratories to deliver highly sensitive and specific analytical methods for detection.

Monitoring Prenatal Exposure to Buprenorphine and Methadone.

PURPOSE: Buprenorphine and methadone are international gold standards for managing opioid use disorders. Although they are efficacious in treating opioid dependence, buprenorphine and methadone present risks, especially during pregnancy, causing neonatal abstinence syndrome and adverse obstetrical outcomes. Buprenorphine and methadone are also abused during pregnancy, and identifying their use is important to limit unprescribed prenatal exposure. Previous studies have suggested that concentrations of buprenorphine, but not methadone markers in unconventional matrices may predict child outcomes, although currently only limited data exist. We reviewed the literature on concentrations of buprenorphine, methadone, and their metabolites in unconventional matrices to improve data interpretation. METHODS: A literature search was conducted using scientific databases (PubMed, Scopus, Web of Science, and reports from international institutions) to review published articles on buprenorphine and methadone monitoring during pregnancy. RESULTS: Buprenorphine and methadone and their metabolites were quantified in the meconium, umbilical cord, placenta, and maternal and neonatal hair. Methadone concentrations in the meconium and hair were typically higher than those in other matrices, although the concentrations in the placenta and umbilical cord were more suitable for predicting neonatal outcomes. Buprenorphine concentrations were lower and required sensitive instrumentation, as measuring buprenorphine glucuronidated metabolites is critical to predict neonatal outcomes. CONCLUSIONS: Unconventional matrices are good alternatives to conventional ones for monitoring drug exposure during pregnancy. However, data are currently scarce on buprenorphine and methadone during pregnancy to accurately interpret their concentrations. Clinical studies should be conducted with larger cohorts, considering confounding factors such as illicit drug co-exposure.

Pyrrolidinyl Synthetic Cathinones α-PHP and 4F-α-PVP Metabolite Profiling Using Human Hepatocyte Incubations.

For more than ten years, new synthetic cathinones (SCs) mimicking the effects of controlled cocaine-like stimulants have flooded the illegal drug market, causing numerous intoxications and fatalities. There are often no data on the pharmacokinetics of these substances when they first emerge onto the market. However, the detection of SC metabolites is often critical in order to prove consumption in clinical and forensic settings. In this research, the metabolite profile of two pyrrolidinyl SCs, α-pyrrolidinohexaphenone (α-PHP) and 4''-fluoro-α-pyrrolidinovalerophenone (4F-α-PVP), were characterized to identify optimal intake markers. Experiments were conducted using pooled human hepatocyte incubations followed by liquid chromatography-high-resolution tandem mass spectrometry and data-mining software. We suggest α-PHP dihydroxy-pyrrolidinyl, α-PHP hexanol, α-PHP 2'-keto-pyrrolidinyl-hexanol, and α-PHP 2'-keto-pyrrolidinyl as markers of α-PHP use, and 4F-α-PVP dihydroxy-pyrrolidinyl, 4F-α-PVP hexanol, 4F-α-PVP 2'-keto-pyrrolidinyl-hexanol, and 4F-α-PVP 2'-keto-pyrrolidinyl as markers of 4F-α-PVP use. These results represent the first data available on 4F-α-PVP metabolism. The metabolic fate of α-PHP was previously studied using human liver microsomes and urine samples from α-PHP users. We identified an additional major metabolite (α-PHP dihydroxy-pyrrolidinyl) that might be crucial for documenting exposure to α-PHP. Further experiments with suitable analytical standards, which are yet to be synthesized, and authentic specimens should be conducted to confirm these results.

A novel, simultaneous extraction of FAEE and EtG from meconium and analysis by LC-MS/MS.

Fatty acid ethyl esters (FAEEs) and ethyl-glucuronide (EtG) in meconium have been widely studied as biomarkers of maternal alcohol consumption during pregnancy. Many analytical approaches have been proposed for their analysis, mostly consisting of separated extraction procedures requiring the use of two meconium aliquots. This study aimed to validate a new analytical procedure for the simultaneous extraction of FAEEs and EtG from a meconium aliquot through a single solid-phase extraction (SPE) applied to 242 anonymized samples of meconium. Targeted FAEEs were: ethyl-myristate (Myr), ethyl-palmitate (Pal), ethyl-oleate (Ole) and ethyl-stearate (Ste). Two hundred milligrams of meconium was sonicated with acetonitrile, and a single SPE performed by means of aminopropyl columns. FAEEs were eluted with hexane, followed by EtG elution with water. Both the mixtures were dried, recovered, and analyzed by liquid chromatography-tandem mass spectrometry using C8 (FAEEs) and C18 (EtG) columns. Transitions were: m/z 257 → 57,88, Myr; m/z 262 → 57,88, Myr-d5; m/z 285 → 57, 72, Pal; m/z 290 → 57,258, Pal-d5; m/z 311 → 72,114, Ole; m/z 316 → 72,265, Ole-d5; m/z 257 → 57,72 Ste; m/z 318 → 57,286, Ste-d5; m/z 221 → 75,85, EtG; m/z 226 → 75,85, EtG-d5. Lower limit of quantification range was 10-15 ng/g for FAEEs and 10 ng/g for EtG. Linearity was evaluated for different concentration ranges; the mean coefficients of determination (R (2)) were above 0.9961. Precision and accuracy for FAEEs and EtG were consistently ≤20 % and ±20 %, respectively. Ion suppression was observed for all the analytes. Matrix effect did not significantly affect the analyses. Recovery efficiency was 93 % for EtG and 75-85 % for FAEEs.

Hepatotoxicity Induced by "the 3Ks": Kava, Kratom and Khat.

The 3Ks (kava, kratom and khat) are herbals that can potentially induce liver injuries. On the one hand, growing controversial data have been reported about the hepatotoxicity of kratom, while, on the other hand, even though kava and khat hepatotoxicity has been investigated, the hepatotoxic effects are still not clear. Chronic recreational use of kratom has been associated with rare instances of acute liver injury. Several studies and case reports have suggested that khat is hepatotoxic, leading to deranged liver enzymes and also histopathological evidence of acute hepatocellular degeneration. Numerous reports of severe hepatotoxicity potentially induced by kava have also been highlighted, both in the USA and Europe. The aim of this review is to focus on the different patterns and the mechanisms of hepatotoxicity induced by "the 3Ks", while trying to clarify the numerous aspects that still need to be addressed.

GHB pharmacology and toxicology: acute intoxication, concentrations in blood and urine in forensic cases and treatment of the withdrawal syndrome.

The illicit recreational drug of abuse, γ-hydroxybutyrate (GHB) is a potent central nervous system depressant and is often encountered during forensic investigations of living and deceased persons. The sodium salt of GHB is registered as a therapeutic agent (Xyrem®), approved in some countries for the treatment of narcolepsy-associated cataplexy and (Alcover®) is an adjuvant medication for detoxification and withdrawal in alcoholics. Trace amounts of GHB are produced endogenously (0.5-1.0 mg/L) in various tissues, including the brain, where it functions as both a precursor and a metabolite of the major inhibitory neurotransmitter γ-aminobutyric acid (GABA). Available information indicates that GHB serves as a neurotransmitter or neuromodulator in the GABAergic system, especially via binding to the GABA-B receptor subtype. Although GHB is listed as a controlled substance in many countries abuse still continues, owing to the availability of precursor drugs, γ-butyrolactone (GBL) and 1,4-butanediol (BD), which are not regulated. After ingestion both GBL and BD are rapidly converted into GHB (t½ ~1 min). The Cmax occurs after 20-40 min and GHB is then eliminated from plasma with a half-life of 30-50 min. Only about 1-5% of the dose of GHB is recoverable in urine and the window of detection is relatively short (3-10 h). This calls for expeditious sampling when evidence of drug use and/or abuse is required in forensic casework. The recreational dose of GHB is not easy to estimate and a concentration in plasma of ~100 mg/L produces euphoria and disinhibition, whereas 500 mg/L might cause death from cardiorespiratory depression. Effective antidotes to reverse the sedative and intoxicating effects of GHB do not exist. The poisoned patients require supportive care, vital signs should be monitored and the airways kept clear in case of emesis. After prolonged regular use of GHB tolerance and dependence develop and abrupt cessation of drug use leads to unpleasant withdrawal symptoms. There is no evidence-based protocol available to deal with GHB withdrawal, apart from administering benzodiazepines.

Anabolic Androgenic Steroid (AAS) related deaths: autoptic, histopathological and toxicological findings.

Anabolic androgenic steroids (AASs) represent a large group of synthetic derivatives of testosterone, produced to maximize anabolic effects and minimize the androgenic ones. AAS can be administered orally, parenterally by intramuscular injection and transdermally. Androgens act by binding to the nuclear androgen receptor (AR) in the cytoplasm and then translocate into the nucleus. This binding results in sequential conformational changes of the receptor affecting the interaction between receptor and protein, and receptor and DNA. Skeletal muscle can be considered as the main target tissue for the anabolic effects of AAS, which are mediated by ARs which after exposure to AASs are up-regulated and their number increases with body building. Therefore, AASs determine an increase in muscle size as a consequence of a dose-dependent hypertrophy resulting in an increase of the cross-sectional areas of both type I and type II muscle fibers and myonuclear domains. Moreover, it has been reported that AASs can increase tolerance to exercise by making the muscles more capable to overload therefore shielding them from muscle fiber damage and improving the level of protein synthesis during recovery. Despite some therapeutic use of AASs, there is also wide abuse among athletes especially bodybuilders in order to improve their performances and to increase muscle growth and lean body mass, taking into account the significant anabolic effects of these drugs. The prolonged misuse and abuse of AASs can determine several adverse effects, some of which may be even fatal especially on the cardiovascular system because they may increase the risk of sudden cardiac death (SCD), myocardial infarction, altered serum lipoproteins, and cardiac hypertrophy. The aim of this review is to focus on deaths related to AAS abuse, trying to evaluate the autoptic, histopathological and toxicological findings in order to investigate the pathophysiological mechanism that underlines this type of death, which is still obscure in several aspects. The review of the literature allowed us to identify 19 fatal cases between 1990 and 2012, in which the autopsy excluded in all cases, extracardiac causes of death.

Smart drugs and synthetic androgens for cognitive and physical enhancement: revolving doors of cosmetic neurology.

Cognitive enhancement can be defined as the use of drugs and/or other means with the aim to improve the cognitive functions of healthy subjects in particular memory, attention, creativity and intelligence in the absence of any medical indication. Currently, it represents one of the most debated topics in the neuroscience community. Human beings always wanted to use substances to improve their cognitive functions, from the use of hallucinogens in ancient civilizations in an attempt to allow them to better communicate with their gods, to the widespread use of caffeine under various forms (energy drinks, tablets, etc.), to the more recent development of drugs such as stimulants and glutamate activators. In the last ten years, increasing attention has been given to the use of cognitive enhancers, but up to now there is still only a limited amount of information concerning the use, effect and functioning of cognitive enhancement in daily life on healthy subjects. The first aim of this paper was to review current trends in the misuse of smart drugs (also known as Nootropics) presently available on the market focusing in detail on methylphenidate, trying to evaluate the potential risk in healthy individuals, especially teenagers and young adults. Moreover, the authors have explored the issue of cognitive enhancement compared to the use of Anabolic Androgenic Steroids (AAS) in sports. Finally, a brief overview of the ethical considerations surrounding human enhancement has been examined.

The impact of nandrolone decanoate on the central nervous system.

Nandrolone is included in the class II of anabolic androgenic steroids (AAS) which is composed of 19-nor-testosterone-derivates. In general, AAS is a broad and rapidly increasing group of synthetic androgens used both clinically and illicitly. AAS in general and nandrolone decanoate (ND) in particular have been associated with several behavioral disorders. The purpose of this review is to summarize the literature concerning studies dealing with ND exposure on animal models, mostly rats that mimic human abuse systems (i.e. supraphysiological doses). We have focused in particular on researches that have investigated how ND alters the function and expression of neuronal signaling molecules that underlie behavior, anxiety, aggression, learning and memory, reproductive behaviors, locomotion and reward.

Enhancing the sensitivity of the LC-MS/MS detection of propofol in urine and blood by azo-coupling derivatization.

Propofol is a low-polarity, volatile molecule that is difficult for an electrospray ion source (ESI) to ionize in either negative ion mode (NIM) or positive ion mode (PIM), which hampers its detection via liquid chromatography-mass spectrometry. The aim of the present study was to use a new derivatization agent to improve ionization efficiency and to develop an efficient liquid chromatography-multiple mass spectrometry (LC-MS/MS) determination of propofol in urine and blood, taking advantage of an electrophilic aromatic substitution. An azo-coupling reaction with a diazonium salt from aniline was performed to introduce a protonation site into the molecule. The diazonium salt was generated by aniline in water solution by HCl and sodium nitrite; derivatization was achieved by stirring a mixture of the diazonium salt and propofol in sodium hydroxide solution for 30 min below 5 °C. A liquid-liquid extraction with dichloromethane and ethyl acetate was performed to obtain the azo derivative (molecular composition: C18H22ON2; molecular weight: 282 Da) in high yield. The compound provided very high ionization yields in both PIM and NIM ESI, and the protonated or deprotonated molecule gave intense signals. The transitions m/z 283 → 77, 241 and m/z 281 → 176, 161 were chosen for the PIM and NIM, respectively, in order to develop quantitative methods of detecting propofol in urine and blood via triple-quadrupole LC-MS/MS. These methods proved to be highly sensitive, with limits of quantification of 0.4 pg/mL and 0.1 ng/mL obtained in the NIM when analyzing 1 mL of urine and 100 µL of blood, respectively.

3-CMC, 4-CMC, and 4-BMC Human Metabolic Profiling: New Major Pathways to Document Consumption of Methcathinone Analogues?

Synthetic cathinones represent one of the largest and most abused new psychoactive substance classes, and have been involved in numerous intoxications and fatalities worldwide. Methcathinone analogues like 3-methylmethcathinone (3-MMC), 3-chloromethcathinone (3-CMC), and 4-CMC currently constitute most of synthetic cathinone seizures in Europe. Documenting their consumption in clinical/forensic casework is therefore essential to tackle this trend. Targeting metabolite markers is a go-to to document consumption in analytical toxicology, and metabolite profiling is crucial to support investigations. We sought to identify 3-CMC, 4-CMC, and 4-bromomethcathinone (4-BMC) human metabolites. The substances were incubated with human hepatocytes; incubates were screened by liquid chromatography-high-resolution tandem mass spectrometry and data were mined with Compound Discoverer (Themo Scientific). 3-CMC-positive blood, urine, and oral fluid and 4-CMC-positive urine and saliva from clinical/forensic casework were analyzed. Analyses were supported by metabolite predictions with GLORYx freeware. Twelve, ten, and ten metabolites were identified for 3-CMC, 4-CMC, and 4-BMC, respectively, with similar transformations occurring for the three cathinones. Major reactions included ketoreduction and N-demethylation. Surprisingly, predominant metabolites were produced by combination of N-demethylation and ω-carboxylation (main metabolite in 3-CMC-positive urine), and combination of ß-ketoreduction, oxidative deamination, and O-glucuronidation (main metabolite in 4-CMC-positive urine). These latter metabolites were detected in negative-ionization mode only and their non-conjugated form was not detected after glucuronide hydrolysis; this metabolic pathway was never reported for any methcathinone analogue susceptible to undergo the same transformations. These results support the need for comprehensive screening strategies in metabolite identification studies, to avoid overlooking significant metabolites and major markers of consumption.

ß'-Phenylfentanyl Metabolism in Primary Human Hepatocyte Incubations: Identification of Potential Biomarkers of Exposure in Clinical and Forensic Toxicology.

From 2014 onwards, illicit fentanyl and analogues have caused numerous intoxications and fatalities worldwide, impacting the demographics of opioid-related overdoses. The identification of cases involving fentanyl analogues is crucial in clinical and forensic settings to treat patients, elucidate intoxications, address drug use disorders and tackle drug trends. However, in analytical toxicology, the concentration of fentanyl analogues in biological matrices is low, making their detection challenging. Therefore, the identification of specific metabolite biomarkers is often required to document consumption. ß'-Phenylfentanyl (N-phenyl-N-[1-(2-phenylethyl)-4-piperidinyl]-benzenepropanamide) is a fentanyl analogue that was first detected in Sweden in 2017 and has recently reemerged onto the American illicit drug market. There is little data available on ß'-phenylfentanyl effects and toxicokinetics and its metabolism is yet to be studied. We aimed to investigate ß'-phenylfentanyl human metabolism to identify potential biomarkers of use. To assist in ß'-phenylfentanyl metabolite identification, a list of putative reactions was generated using in silico predictions with GLORYx freeware. ß'-phenylfentanyl was incubated with cryopreserved 10-donor-pooled human hepatocytes, analyses were performed by liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS-MS) and data were processed using a partially automated targeted/untargeted approach with Compound Discoverer. We identified 26 metabolites produced by N-dealkylation, oxidation, hydroxylation, O-glucuronidation, O-methylation and combinations thereof. We suggest ß'-phenylnorfentanyl (N-phenyl-N-4-piperidinyl-benzenepropanamide) and further metabolites 1-oxo-N-phenyl-N-4-piperidinyl-benzenepropanamide and 1-hydroxy-N-phenyl-N-4-piperidinyl-benzenepropanamide as major biomarkers of ß'-phenylfentanyl use. In silico predictions were mostly wrong, and ß'-phenylfentanyl metabolic fate substantially differed from that of a closely related analogue incubated in the same conditions, highlighting the value of the experimental assessment of new psychoactive substance human metabolism. In vivo data are necessary to confirm the present results. However, the present results may be necessary to help analytical toxicologists identify ß'-phenylfentanyl-positive cases to provide authentic samples.

Fourth Generation of Synthetic Cannabinoid Receptor Agonists: A Review on the Latest Insights.

BACKGROUND: Over the past few years, an emerging number of new psychoactive substances (NPSs) entered the illicit market. NPSs are designed to resemble the effects of classical drugs of abuse, reinforcing their effects and duration. Among the most abused NPS, synthetic cannabinoids are cannabinoid receptor agonists (SCRAs) that mimic the effect of the main psychotropic phytocannabinoid Δ9-tetrahydrocannabinol (THC). METHODS: We herein reviewed the international literature to provide available information on the newest SCRAs generation. RESULTS: Compared to the previous SCRAs generations, the structures of the last generation result in increased affinity for and efficacy at cannabinoid CB1 receptors, which are thought to be mainly responsible for the psychoactive effects of THC and its analogues. Accordingly, these more potent cannabimimetic effects may increase the number of adverse reactions such as neurological disorders (e.g., psychosis, agitation, irritability, paranoia, confusion, and anxiety), psychiatric episodes (e.g., hallucinations, delusions, self-harm), other physical conditions (e.g., tachycardia, hypertension, arrhythmia, chest pain, nausea, vomiting, and fever) and deaths. In the last decade, more than a hundred SCRAs from different chemical classes emerged on the illicit web market. SCRAs have been thoroughly studied: they were physico-chemically characterized, and pharmaco-toxicological characteristics were investigated. The last SCRAs generations include increasingly potent and toxic compounds, posing a potential health threat to consumers. CONCLUSION: From November 2017 to February 2021, at least 20 new "fourth-generation" SCRAs were formally reported to international drug agencies. Our understanding of the neurotoxicity of these compounds is still limited due to the lack of global data, but their potency and their toxicity are likely higher than those of the previous generations.

3F-α-pyrrolydinovalerophenone (3F-α-PVP) in vitro human metabolism: Multiple in silico predictions to assist in LC-HRMS/MS analysis and targeted/untargeted data mining.

Synthetic cathinones (SCs) constitute a heterogenous class of new psychoactive substances (NPS), structurally related to cathinone. SCs represent the widest NPS class, second to synthetic cannabinoids, accounting for approximately 160 different analogues with substitution at the phenyl group, the amine group, or the alkyl chain. In 2020, α-pyrrolidonophenone analogues were the most trafficked SCs, and were involved in many fatalities and intoxication cases. In particular, 3F-α-pyrrolidinovalerophenone (3F-α-PVP) was the cause of the highest number of SC-related fatal intoxications in Sweden in 2018. Minor structural modifications are used to avoid legal controls and analytical detection, but may also induce different toxicological profile. Therefore, the identification of specific markers of consumption is essential to discriminate SCs in clinical and forensic toxicology. In this study, we assessed 3F-α-PVP metabolic profile. 3F-α-PVP was incubated with 10-donor-pooled human hepatocytes, LC-HRMS/MS analysis, and software-assisted data mining. This well-established workflow was completed by in silico metabolite predictions using three different freeware. Ten metabolites were identified after 3 h incubation, including hydrogenated, hydroxylated, oxidated, and N-dealkylated metabolites. A total of 51 phase I and II metabolites were predicted, among which 7 were detected in the incubations. We suggest 3F-α-PVP N-butanoic acid, 3F-α-PVP pentanol, and 3F-α-PVP 2-ketopyrrolidinyl-pentanol as specific biomarkers of 3F-α-PVP consumption. This is the first time that an N-ethanoic acid is detected in the metabolic pathway of a pyrrolidine SC, demonstrating the importance of a dual targeted/untargeted data mining strategy.