Comprehensive Monitoring of Psychoactive Substances in Psychiatric Patients Using Liquid Chromatography-High-Resolution Mass Spectrometry: A Key Tool for Treatment Planning and Understanding Consumption Patterns in Rome, Italy.

BACKGROUND: The comprehensive monitoring of licit and illicit drug consumption plays a crucial role in understanding the complexities of patient conditions and designing effective treatment strategies. In this study, the prevalence of psychoactive prescription drugs, classical illicit drugs, and new psychoactive substances (NPS) were objectively assessed in individuals diagnosed with drug-related psychiatric disorders or episodes. METHODS: Blood, urine, and hair samples were collected from psychiatric patients admitted to the Mental Health Department and Drug Addiction Service of the North Rome Local Health Authority with declared or suspected psychoactive drug use. Comprehensive drug screening was conducted for all samples using ultra-high-performance liquid chromatography-high-resolution mass spectrometry. RESULTS: A total of 71 blood and urine and 50 hair samples were analyzed to confirm the suitability of the ultra-high-performance liquid chromatography-high-resolution mass spectrometry method for the study purposes. The main substances found in blood and urine were antipsychotics (71.8% and 66.2%) and benzodiazepines (62.0% and 59.2%), respectively, whereas cocaine (84.0%) and antipsychotics (74.0%) was more evident in hair. Z-drugs were detected in blood (7.0%), urine (5.6%), and hair (24%) samples; amphetamines were mainly detected in hair samples (14.0%). Synthetic cathinones were the most frequently detected NPS in hair specimens (8.0%), whereas synthetic cannabinoids were mainly found in blood samples (11.3%). These analyses showed that patients were polydrug users (77.5% detected in blood and urine, and 94.0% in hair). CONCLUSIONS: Comprehensive screening enabled the assessment of past, recent, and actual consumption of psychoactive substances, including licit and illicit drugs and NPS, by psychiatric patients. A thorough understanding of substance consumption patterns can enhance therapeutic interventions and management of psychiatric disorders associated with psychoactive substance use.

New Psychoactive Substances Toxicity: A Systematic Review of Acute and Chronic Psychiatric Effects.

New psychoactive substances (NPSs) are a heterogenous group of psychotropic molecules and diverted pharmaceutical drugs sold worldwide as legal substitutes for controlled drugs. The psychiatric consequences of NPS use are relatively unknown, although evidence of related psychotic symptoms has been described in the literature. We sought to summarize the available evidence on NPS-related psychiatric disorders, to facilitate the interpretation of the molecular mechanism underlying their specific pathologies. A literature search of Scopus, PubMed and Google Scholar was conducted including studies published between 2013 and 2024, in which a correlation between NPS consumption and psychiatric symptoms was reported. Furthermore, the short- and long-term psychopathological effects were included. The literature search resulted in 109 NPS-related intoxication cases in which acute or chronic psychiatric symptoms were reported, mostly related to synthetic cannabinoids, followed by synthetic cathinones, hallucinogens, natural NPSs and stimulants. The most common acute symptoms were hallucinations, aggressiveness, and psychotic and bizarre behavior, related to the molecular disbalance of neurotransmitters in the central nervous systems, with different mechanisms. The lack of clear diagnostic criteria and toxicological analyses has resulted in crucial complications in psychiatric diagnoses related to NPS intoxication. Hence, the implementation of toxicological screening procedures in emergency rooms, including the main NPS classes, should support the diagnosis of acute intoxication and its proper therapeutic treatment. Finally, proper follow-up should be implemented to assess the chronic sequelae.

QuEChERS Extraction and Simultaneous Quantification in GC-MS/MS of Hexahydrocannabinol Epimers and Their Metabolites in Whole Blood, Urine, and Oral Fluid.

Recently, hexahydrocannabinol (HHC) was posed under strict control in Europe due to the increasing HHC-containing material seizures. The lack of analytical methods in clinical laboratories to detect HHC and its metabolites in biological matrices may result in related intoxication underreporting. We developed and validated a comprehensive GC-MS/MS method to quantify 9(R)-HHC, 9(S)-HHC, 9αOH-HHC, 9ßOH-HHC, 8(R)OH-9(R)-HHC, 8(S)OH-9(S)HHC, 11OH-9(R)HHC, 11OH-9(S)HHC, 11nor-carboxy-9(R)-HHC, and 11nor-carboxy-9(S)-HHC in whole blood, urine, and oral fluid. A novel QuEChERS extraction protocol was optimized selecting the best extraction conditions suitable for all the three matrices. Urine and blood were incubated with ß-glucuronidase at 60 °C for 2 h. QuEChERS extraction was developed assessing different ratios of Na2SO4:NaCl (4:1, 2:1, 1:1, w/w) to be added to 200 µL of any matrix added with acetonitrile. The chromatographic separation was achieved on a 7890B GC with an HP-5ms column, (30 m, 0.25 mm × 0.25 µm) in 12.50 min. The analytes were detected with a triple-quadrupole mass spectrometer in the MRM mode. The method was fully validated following OSAC guidelines. The method showed good validation parameters in all the matrices. The method was applied to ten real samples of whole blood (n = 4), urine (n = 3), and oral fluid (n = 3). 9(R)-HHC was the prevalent epimer in all the samples (9(R)/9(S) = 2.26). As reported, hydroxylated metabolites are proposed as urinary biomarkers, while carboxylated metabolites are hematic biomarkers. Furthermore, 8(R)OH-9(R)HHC was confirmed as the most abundant metabolite in all urine samples.

Molecular Insights of New Psychoactive Substances (NPS) 2.0.

The New Psychoactive Substances (NPS) phenomenon represents an ever-changing global issue, with a number of new molecules entering the illicit market every year in response to international banning laws [...].

Sweat Testing for the Detection of Methylone after Controlled Administrations in Humans.

The aim of this study was to determine the excretion of methylone and its metabolites in sweat following the ingestion of increasing controlled doses of 50, 100, 150 and 200 mg of methylone to twelve healthy volunteers involved in a clinical trial. Methylone and its metabolites 4-hydroxy-3-methoxy-N-methylcathinone (HMMC) and 3,4-methylenedioxycathinone (MDC) were analyzed in sweat patches by liquid chromatography-tandem mass spectrometry. Methylone and MDC were detected in sweat at 2 h and reached their highest accumulation (Cmax) at 24 h after the administration of 50, 100, 150 and 200 mg doses. In contrast, HMMC was not detectable at any time interval after each dose. Sweat proved to be a suitable matrix for methylone and its metabolites' determination in clinical and toxicological studies, providing a concentration that reveals recent drug consumption.

Molecular Insights and Clinical Outcomes of Drugs of Abuse Adulteration: New Trends and New Psychoactive Substances.

Adulteration is a well-known practice of drug manufacturers at different stages of drug production. The intentional addition of active ingredients to adulterate the primary drug may enhance or mask pharmacological effects or may produce more potent drugs to increase the number of available doses and the dealer's profit. Adulterants found in different drugs change over time in response to different factors. A systematic literature search in PubMed and Scopus databases and official international organizations' websites according to PRISMA guidelines was performed. A total of 724 studies were initially screened, with 145 articles from PubMed and 462 from Scopus excluded according to the criteria described in the Method Section. The remaining 117 records were further assessed for eligibility to exclude articles without sufficient data. Finally, 79 studies were classified as "non-biological" (n = 35) or "biological" (n = 35 case reports; n = 9 case series) according to the samples investigated. Although the seized samples analyses revealed the presence of well-established adulterants such as levamisole for cocaine or paracetamol/acetaminophen for heroin, the reported data disclosed new adulteration practices, such as the use of NPS as cutting agents for classic drugs of abuse and other NPS. For example, heroin adulterated with synthetic cannabinoids or cocaine adulterated with fentanyl/fentalogues raised particular concern. Notably, adulterants play a role in some adverse effects commonly associated with the primary drug, such as levamisole-adulterated cocaine that may induce vasculitis via an autoimmune process. It is essential to constantly monitor adulterants due to their changing availability that may threaten drug consumers' health.

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.

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.

Development and validation of fast UHPLC-MS/MS screening method for 87 NPS and 32 other drugs of abuse in hair and nails: application to real cases.

Interest on keratinized matrix analysis for clinical and forensic purposes has been recently grown due to the wide temporary detection window for psychotropic and toxic substances entrapped after repeated consumption. The aim of this study was the development and full validation of an UHPLC-MS/MS screening method to quantify 119 molecules among most abused classic drugs and new psychoactive substances in hair and in nails, to assess the polyconsumption. Twenty-five milligrams of hair or nail samples, added with the internal standard mixture, were cut and incubated with 500 µL M3® buffer reagent at controlled temperature. After cooling, 1 µL supernatant was injected in the chromatographic system equipped with an Oasis HLB column. After the 10 min chromatographic separation through a gradient mobile phase (aqueous ammonium formate, phase A; acetonitrile, phase B), the target compounds were detected in multiple reaction monitoring mode. The method was linear (r2 always better than 0.99) in a calibration range of LOQ 20000 pg compound for milligram hair and of LOQ 1000 pg compound per milligram nail. Process efficiency of analytes under investigation was always better than 65% and no significant ion suppression due to matrix effect was observed. Intra-assay and inter-assay precision and accuracy were always better than 15%. The applicability and trueness of the method were examined by analysing real samples of hair and nail from users of psychoactive drugs in recreational contexts. Both classic drugs and new psychoactive substances could be determined as result of single or repeated use and accumulation in keratin matrices. Graphical abstract.

"Tranq-dope": The first fatal intoxication due to xylazine-adulterated heroin in Italy.

BACKGROUND AND AIMS: The number of xylazine-involved overdose deaths tremendously increased from 2019 onwards in the US. This is due to the "tranq-dope" trend consisting in mixing opioids with the sedative to reduce drug manufacturing costs and enhance their effects. In this study, we report the first fatality involving xylazine-adulterated heroin in the EU. MATERIALS AND METHODS: The subject was a 33-year-old Caucasian male with a documented history of drug abuse who was found dead in a public area with puncture marks at the elbow. Peripheral blood and urine were collected at the autopsy and analyzed by liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS/MS) after protein precipitation. RESULTS: 6-Monoacetylmorphine, total/free morphine, and codeine blood concentrations of 20.3, 236/105, and 38.3 ng/mL, respectively, indicated recent heroin consumption. Methadone blood concentration was below 10 ng/mL. Alprazolam, nordiazepam, and flurazepam blood concentrations were 23.9, 61.4, and 55.0 ng/mL, respectively. Benzoylecgonine blood concentration was below 5 ng/mL. Xylazine blood and urine concentrations were 105 and 72.6 ng/mL, respectively. CONCLUSION: The combination of central nervous system depressants, i.e., opioids, benzodiazepines, and xylazine, was the principal cause of death by cardiorespiratory failure. The case was promptly reported to the UE Early Warning System on drugs.

Recent insights in the correlation between social media use, personality traits and exercise addiction: a literature review.

Introduction and aim: The excessive involvement in physical activity without stopping in between sessions despite injuries, the continuous thinking to exercise feeling insane thoughts and experiencing withdrawal symptoms are all characteristics of the Exercise Addiction (EA), an addictive behavior. While the primary exercise addiction is directly caused by compulsive exercise, many studies highlighted the relationship between Eating Disorders (ED) and EA, defining the secondary EA. The correlation between EA, social media use (SMU) and other individual traits remains a relatively underexplored domain. Therefore, this review aimed to examine the latest evidence on the relationship between EA, SMU, and some personality traits such as perfectionism and body image. Methods: Electronic databases including PubMed, Medline, PsycARTICLES, Embase, Web of Science were searched from January 2019 to October 2023, following the PRISMA guidelines. Results: A total of 15 articles were examined and consolidated in this review. EA was found to be related to different individual traits such perfectionism, body dissatisfaction, depression, obsessive-compulsive personality disorders. While controversial results were found regarding the relationship between EA and SMU. Conclusion: The interaction between mental health, exercise addiction and social media use is complex. Excessive engagement in these latter may result in negative mental health consequences despite their potential benefits. Understanding individual differences and developing effective interventions is crucial to promoting healthy habits and mitigating the EA risks, ultimately enhancing mental well-being. Further research should focus on the identification of risks and protective factors with the eventual aim of developing and implementing effective prevention strategies.

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.

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.

Hexahydrocannabinol Pharmacology, Toxicology, and Analysis: The First Evidence for a Recent New Psychoactive Substance.

BACKGROUND: During the last two years, hexahydrocannabinol (HHC), the hydrogenated derivative of tetrahydrocannabinol has been freely sold by internet websites as a "legal" replacement to THC and cannabis in a range of highly attractive branded and unbranded products, some of which are sold as "legal highs". Potentially, there could be a large demand for HHC products by individuals in Europe and internationally. METHODS: Studies reporting HHC pharmacology, toxicology and analysis were identified from Pubmed and Scopus databases, and official international organizations' websites were considered. RESULTS: HHC showed the effects of the typical cannabinoid on the central nervous system, with lower potency than Δ9-THC. A few studies highlighted that 9(R)-HHC is more potent than 9(S)-HHC. This molecule showed an affinity for cannabinoid receptor CB1 both in vitro and in vivo, suggesting a possible therapeutic effect in several pathologies. However, the affinity for the CB1 receptor suggests a possible addiction potential, inducing the users to misuse it. Since actual intoxication cases have not yet been reported, the HHC harmful potential was not described, probably due to the lack of effective analytical methods to detect HHC in biological matrices. Conversely, different analytical assays were developed and validated to separate HHC epimers in natural and non-natural sources. CONCLUSION: Similarly to other NPS, the HHC represents a cheaper alternative to the controlled Δ9-THC. Its monitoring is a crucial challenge for toxicological and forensic purposes. To this concern, it is essential to further investigate HHC to support health providers in the identification of related intoxications.

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.

The effects of the COVID-19 pandemic on the use of the performance-enhancing drugs.

The coronavirus disease (COVID-19) pandemic has been impacting the whole society in every aspect of the daily life, comprising the sport field. Several restrictive strategies have been implemented by governments in an effort to stem the spread of the disease and salvage public health. Such efforts have severely constrained access to non-essential services, leading to the closure of non-essential points of gathering and business and the enforcement of rigorous social distancing and prolonged lockdowns, in addition to masking and stay-at-home mandates. However necessary, there is no denying that such extremely rigorous, and to most people unprecedented, measures have adversely affected the global economy and the daily lives of everyone of us, including professional and amateur athletes (1). The most important sport events were postponed or cancelled, including the 2020 Tokyo Olympic Games. But how was the phenomenon of performance-enhancing drug (PED) use impacted and how was the most concerning issue affecting the integrity of sport affected by the pandemic control restrictions? The World Anti-doping Agency (WADA) was established in 1999, whereas its code was implemented in 2004 in order to articulate and enforce doping control initiatives and provide educational strategies aimed at preventing PED use (2). Nonetheless, it is worth noting that the prevalence of PED use among athletes is mostly unchanged since the foundation of WADA. Unfortunately, the use of the performance-enhancing drugs is not limited to athletic performances, but it concerns other settings as well. Nowadays, several strategies for doping control are adopted such as education, deterrence, detection, enforcement and rule of law (3), but the most important anti-dissemination strategy is constituted by information campaigns, especially addressed to youngsters, meant to raise awareness as to the serious health risks involved in PED use. Currently, the primary drivers of anabolic androgenic steroids (ASA) use are 1) the determination to improve performances and prevail no matter what the cost may be; 2) the economic benefits, popularity and fame; 3) greater stamina and resistance. This public health issue has raised particular concerns due to the recent ASA market developments, which is somewhat similar to the illicit market of narcotic drugs. Moreover, it has to be considered that the higher stress and psychosocial condition related to pandemic social restrictions has fueled and exacerbated substance use disorders (4). The prevalence of doping in sport causes unfairness and damages the very fabric of our society, especially insofar as it involves children and young adults who look up to athletes as role models. In this concern, the impact of the COVD-19 pandemic may have led to substantial modifications in substance use patterns and an increased risk of substitution, adulteration, contamination, and dilution with a potentially harmful substance (5, 6). During the COVID-19 lockdown, WADA and stakeholders suspended or scaled down doping control programs, testing and other activities. As a consequence, athletes have seen the unexpected opportunity to misuse AAS without the possible risk of testing positive (7). This has been controversial, considering the measures taken by governments to flatten the pandemic curve in order to safeguard public health. Indeed, all the technologies implemented for teleworking, such as teaching students on-line, telehealth applications, prescriptions and referrals, and treating patients in hospitals/care homes via video links can also be applied to enhance and uphold sport integrity. Conversely, anti-doping testing for professional competitive athletes has increased, due to the lockdown raising suspicion about doping opportunities. The U.S. Anti-doping Agency has put in place novel measures to combat the lack of anti-doping testing during the pandemic: these include a "in-home self-test" that requires athletes to provide urine and small blood samples at home to be tested in the anti-doping laboratory, under supervision provided by video-conference (8). As such, reports from forensic science and toxicology laboratories are crucial for the early detection and response to such events. Furthermore, toxicology laboratories should assure their continue effort in providing new methods and technologies designed to tackle the consumption of illicit substances and to monitor the constantly changing illegal drug markets (9). The most recent WADA code revision has certainly brought about important progress in the ongoing fight against PED abuse. Indeed, it has introduced the possibility to store the samples for 10 years after the first analysis, maintaining the same legal value if re-tested and use for prosecution purposes (10). In that regard, the prospect of re-testing the same sample with newly developed analytical methods based on innovative technologies may represent a strong deterrent for doping users, since anti-doping research rapidly evolves (6), largely by implementing the same approaches used to fight new psychoactive substances (NPS) use (11,12). It is worth noting that the NPS phenomenon bears several similarities with doping, especially due to the constant emergence of new substances and methods aimed at circumventing current legal restrictions. In Italy, the National Antidoping Organization (NADO-Italia) is in charge of guaranteeing compliance with WADA rules and the transposition of the List of Prohibited Substances and Methods. However, the gap between elite athletes and amateur athletes is still broad and unaddressed, since non-professional sport competitions are not adequately overseen, and neither are the competing athletes . This difference may give rise to an important public health issue, on account of the adverse effects of uncontrolled doping agents consumption. In this concern, the Italian anti-doping law created the "Section of the Technical Health Committee for Supervision and Control on Doping and for Health Protection in Sport Activities", that carries out, among its other tasks, the following activities on amateur sport: 1) updating each year the list of banned substances and practices, adapting it to the WADA list; 2) determining cases, criteria and methodologies for anti-doping controls; 3) promoting research projects and information/training campaigns meant to protect health in sports and tackle doping (13). In conclusion, regarding the highly complex dynamics triggered by the pandemic, new and unexpected challenges have come to the fore in the ongoing fight against substance abuse in its every aspect, such as NPS (14), ASA consumption by amateur athletes, or other substance abuse settings, e.g. driving under the influence of psychotropic substances (15). The current Italian antidoping approach for amateur athletes seems to be a promising strategy to bridge the gap between professional sports and amateur sports. Moreover, youngsters should be thoroughly educated as to the threats posed by such substances, so that they can realize how profoundly and severely drug abuse can affect not only their sport career, but their health and well-being overall.

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.

In silico prediction, LC-HRMS/MS analysis, and targeted/untargeted data-mining workflow for the profiling of phenylfentanyl in vitro metabolites.

Illicit fentanyl and analogues have been involved in many fatalities and cases of intoxication across the United States over the last decade, and are becoming a health concern in Europe. New potent analogues emerge onto the drug market every year to circumvent analytical detection and legislation, and little pharmacological/toxicological data are available when the substances first appear. However, pharmacokinetic data are crucial to determine specific biomarkers of consumption in clinical and forensic settings, considering the low active doses and the rapid metabolism of fentanyl analogues. Phenylfentanyl is a novel analogue that was first detected in seized material in 2017, and little is currently known about this substance and its metabolism. We studied phenylfentanyl metabolic fate using in silico predictions with GLORYx freeware, human hepatocyte incubations, and liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS/MS). We applied a specific targeted/untargeted workflow using data-mining software to allow the rapid and partially automated screening of LC-HRMS/MS raw data. Approximately 90,000 substances were initially individuated after 3-h incubation with hepatocytes, and 115 substances were automatically selected for a manual check by the operators. Finally, 13 metabolites, mostly produced by N-dealkylation, amide hydrolysis, oxidation, and combinations thereof, were identified. We suggest phenylnorfentanyl as the main biological marker of phenylfentanyl use, and we proposed the inclusion of its fragmentation pattern in mzCloud and HighResNPS online libraries. Other major metabolites include N-Phenyl-1-(2-phenylethyl)-4-piperidinamine (4-ANPP), 1-(2-phenylethyl)-4-piperidinol, and other non-specific metabolites. Phase II transformations were infrequent, and the hydrolysis of the biological samples is not required to increase the detection capability of non-conjugated metabolites. The overall workflow is easily adaptable for the metabolite profiling of other novel psychoactive substances.