Detecting novel psychoactive substances around the world.

PURPOSE OF REVIEW: The worldwide spread of novel psychoactive substances (NPS) in the illicit drug market and their continuous increase in number and type, for the purpose of bypassing controlled substance legislation, represents a continuing challenge for forensic scientists, clinicians and enforcement authorities. We aim to provide information regarding the most urgent harms related to NPS consumption in different world regions and the current state of the art for NPS analysis. RECENT FINDINGS: Unfortunately, the identification of NPS in biological samples is controversial, especially when samples are limited, or the drug is promptly and extensively metabolized. This causes a lack of information on their real diffusion in different parts of the world and in different populations. New technologies and instrumental detection of NPS in alternative samples are offering comprehensive information about NPS use. SUMMARY: The lack of detection and underreporting of NPS in biological samples makes it difficult to obtain complete qualitative and quantitative information about NPS prevalence. The most innovative strategies that have been proposed in the last 2 years to assist NPS analysis and possibly facilitate the understanding of the NPS diffusion around the world are presented.

Enhancing breast cancer screening with urinary biomarkers and Random Forest supervised classification: A comprehensive investigation.

OBJECTIVES: Urinary sex hormones are investigated as potential biomarkers for the early detection of breast cancer, aiming to evaluate their relevance and applicability, in combination with supervised machine-learning data analysis, toward the ultimate goal of extensive screening. METHODS: Sex hormones were determined on urine samples collected from 250 post-menopausal women (65 healthy - 185 with breast cancer, recruited among the clinical patients of Candiolo Cancer Institute FPO-IRCCS (Torino, Italy). Two analytical procedures based on UHPLC-MS/HRMS were developed and comprehensively validated to quantify 20 free and conjugated sex hormones from urine samples. The quantitative data were processed by seven machine learning algorithms. The efficiency of the resulting models was compared. RESULTS: Among the tested models aimed to relate urinary estrogen and androgen levels and the occurrence of breast cancer, Random Forest (RF) proved to underscore all the other supervised classification approaches, including Partial Least Squares - Discriminant Analysis (PLS-DA), in terms of effectiveness and robustness. The final optimized model built on only five biomarkers (testosterone-sulphate, alpha-estradiol, 4-methoxyestradiol, DHEA-sulphate, and epitestosterone-sulphate) achieved an approximate 98% diagnostic accuracy on replicated validation sets. To balance the less-represented population of healthy women, a Synthetic Minority Oversampling TEchnique (SMOTE) data oversampling approach was applied. CONCLUSIONS: By means of tunable hyperparameters optimization, the RF algorithm showed great potential for early breast cancer detection, as it provides clear biomarkers ranking and their relative efficiency, allowing to ground the final diagnostic model on a restricted selection five steroid biomarkers only, as desirable for noninvasive tests with wide screening purposes.

Metabolic profile of N-ethylhexedrone, N-ethylpentedrone, and 4-chloromethcathinone in urine samples by UHPLC-QTOF-HRMS.

Forensic laboratories are constantly required to identify new drugs and their metabolites. N-ethylhexedrone (NEH, HEXEN), N-Ethylpentedrone (NEP), and 4-Chloromethcathinone (4-CMC, clephedrone) are synthetic substances structurally related to natural cathinone, alkaloid present in the leaves of the Catha edulis (Khat) plant. These synthetic cathinones (SC) are members of the heterogenous family of new psychoactive substances (NPS) that raised major concerns in scientific and forensic communities over the past years due to their widespread consumption. In this context, we investigated their metabolic profile using of UHPLC-QTOF-HRMS to elucidate the distribution of the parent drug and its metabolites in urine samples over time. Initially, both male and female volunteers were divided into three groups and eight subjects of each group were administered intranasally or orally with one SC (20-40 mg of NEH or NEP intranasal, 100-150 mg of 4-CMC oral). Urine samples were collected at 0-2 and 2-4 or 2-5 h. Urine (50 µL) was diluted 1:2 with acetonitrile/methanol (95:5) and injected into the UHPLC-QTOF-HRMS. Phase-I and phase-II metabolites were identified on the basis of fragmentation patterns and exact masses. Several phase-I and glucuronide-phase-II metabolites were identified in urine samples. Keto group reduction, hydroxylation and dealkylation were the common metabolic pathways identified for all cathinones and the presence of NEH-glucuronide, NEP-glucuronide and 4-CMC-glucuronide was also relevant. Significant is the slower metabolite formation for 4-CMC, which was detected at high concentrations in its original form even 5 h after administration, due to its long half-life and low intrinsic clearance compared to the other SCs. UHPLC-QTOF-HRMS demonstrated a considerable capability to semi-quantify the three synthetic cathinones and identify the target metabolites with high reliability. The introduction of new target compounds improves the efficiency of toxicological screening analysis on real samples and extends the window of detection of the SCs in biological matrices.

Method development for the quantification of nine nitazene analogs and brorphine in Dried Blood Spots utilizing liquid chromatography - tandem mass spectrometry.

The detection of nitazenes in biological fluids is increasingly needed as they are repeatedly reported in intoxication and overdose cases. A simple method for the quantification of low levels of nine nitazene analogs and brorphine in Dried Blood Spots (DBS) was developed and validated. 10 µL of spiked whole blood is deposited on a Capitainer®B card and allowed to dry. The spot is punched out, and extracted with 500 µL methanol:acetonitrile (3:1 v/v) added with 1.5 µL of fentanyl-D5 as the internal standard. After stirring, sonication, and centrifugation of the vial, the solvent is dried under nitrogen, the extract is reconstituted in 30 µL methanol, and 1 µL is injected into a UHPLC-MS/MS instrument. The method validation showed linear calibration in the 1-50 ng/mL range, LOD values ranging between 0.3 ng/mL (isotonitazene) and 0.5 ng/mL (brorphine), average CV% and bias% within 15 % and 10 % for all compounds, respectively. The matrix effect due to blood and filter paper components was within 85-115 % while recovery was between 15-20 %. Stability tests against time and temperature showed no significant variations for storage periods up to 28 days. Room temperature proved to represent the best samples storage conditions. UHPLC-MS/MS proved capable to reliably identify all target analytes at low concentration even in small specimen volumes, as those obtained from DBS cards, which in turn confirmed to be effective and sustainable micro-sampling devices. This procedure improves the efficiency of toxicological testing and provides an innovative approach for the identification of the nitazene class of illicit compounds.

Five cases of unintentional exposure to BZO-4en-POXIZID among nightclub attendees in New York City.

A new class of synthetic cannabinoids called OXIZIDs has emerged in recent years. This class consists of compounds with oxindole cores and hydrazide/hydrazone linker moieties and has often been described as being designed to circumvent a Chinese class-wide ban that was effective as of 1 July 2021. However, through hair testing of nightclub attendees in New York City-a high-risk population for recreational drug use-we have evidence suggesting exposures to an OXIZID called BZO-4en-POXIZID (4en-pentyl MDA-19) prior to the effective ban. Through analysis of 6 cm segmented hair samples from attendees collected in 2021, we detected five cases of exposure. Specifically, we detected a cluster of three cases based on hair samples collected on 20 June 2021, and then two additional cases from samples collected on 16 July 2021. Four of these hair samples were long enough to analyze two 6 cm hair segments (representing approximately two 6-month timeframes) and three of four of these cases tested positive for repeated exposure (for an estimated exposure over 6 months prior to hair collection). All cases included young adult females reporting past-year cannabis use but all tested negative for tetrahydrocannabinol exposure. Three cases also reported past-year use of cocaine, ecstasy, and/or ketamine, and four cases tested positive for exposure to cocaine, 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), methamphetamine and/or eutylone. These subjects were exposed to BZO-4en-POXIZID-likely as an adulterant in other drugs, and these cases are among the first documented cases which occurred approximately half a year before the Chinese legislative ban.

A multivariable analysis delineating hair color, hair dyeing, and hat wearing as predictors of level of cocaine and MDMA detection in human hair samples.

Research suggests that hair color, hair dyeing, and perspiration can bias hair test results regarding drug exposure, but research is needed to examine such associations in a multivariable manner. In this epidemiology study, adults were surveyed entering nightclubs and dance festivals in New York City, and 328 provided hair samples, which were analyzed using ultra-high-performance liquid chromatography-tandem mass spectrometry to determine the level of detection of cocaine and 3,4-methylenedioxymethamphetamine (MDMA). Reporting use was not an inclusion criterion for analysis. We used two-part multivariable models to delineate associations of hair color, past-year hair dyeing, and frequency of past-month hat wearing (which may increase perspiration) in relation to any vs. no detection of cocaine and MDMA as well as level of detection, controlling for hair length, self-reported past-year cocaine/ecstasy/MDMA use, and age, sex, and race/ethnicity. Those reporting having dyed their hair were at increased odds of having any level of cocaine detected (adjusted odds ratio [aOR] = 3.75, 95% CI confidence interval [CI]: 1.85-6.70), and compared to those with brown hair, those with blond(e) hair on average had lower levels of cocaine (ng/mg) detected (beta = -7.97, p = 0.025). Those reporting having dyed their hair were at increased odds of having any level of MDMA detected (aOR = 3.05, 95% CI: 1.44-6.48), and compared to those who reported never wearing a hat, those who reported wearing a hat daily or almost daily on average had lower levels of MDMA (ng/mg) detected (beta = -6.61, p = 0.025). This study demonstrates the importance of using multivariable models to delineate predictors of drug detection.

Trends and correlates of discordant reporting of drug use among nightclub/festival attendees, 2019-2022.

INTRODUCTION: People who attend nightclubs and festivals are known for high prevalence of party drug use, but more research is needed on underreporting in this population, in part because unintentional drug exposure through adulterated drug products is common. We examined the prevalence of drug use in this population, based both on self-reporting and on hair test results, with a focus on the detection of underreported use. METHODS: Adults entering nightclubs and festivals in New York City were asked about past-year drug use in 2019-2022 (n = 1,953), with 328 providing an analyzable hair sample for testing. We compared trends in self-reported drug use, drug positivity, and "corrected" prevalence, adjusting for unreported use, and delineated correlates of testing positive for ketamine and cocaine after not reporting use (discordant reporting). RESULTS: Of the 328 who provided a sample, cocaine and ketamine were the most frequently detected drugs (55.2% [n = 181] and 37.2% [n = 122], respectively), but these were also the two most underreported drugs, with 37.1% (n = 65) and 26.4% (n = 65), respectively, testing positive after not reporting use. Between 2019 and 2022, positivity decreased for cocaine, ketamine, 3,4-methylenedioxy-metamfetamine, and amfetamine, and underreported exposure to cocaine and ketamine also decreased (P < 0.05). Underreporting of the use of these drugs was common, but we also detected underreported exposure to ethylone, fentanyl, 3,4-methylenedioxyamfetamine, metamfetamine, and synthetic cannabinoids. Prevalence of discordant reporting of cocaine use was higher among those testing positive for ketamine exposure (adjusted prevalence ratio = 2.63; 95% CI: 1.48-4.69) and prevalence of discordant reporting of ketamine use was lower post-coronavirus disease caused by the SARS-CoV-2 virus (adjusted prevalence ratio = 0.39; 95% CI: 0.16-0.91) and among those reporting cocaine use (adjusted prevalence ratio = 0.53; 95% CI: 0.32-0.89). DISCUSSION: Underreporting of drug use was common, suggesting the need for researchers to better deduce intentional underreporting versus unknown drug exposure via adulterants. CONCLUSIONS: Researchers should consider both self-report and toxicology results from biological samples when examining trends in use.

Trends in reported and biologically confirmed drug use among people who use ecstasy in the nightclub/festival-attending population, 2016-2022.

Background: Nightclub/festival attendees are a population with high rates of party drug use, but research is needed to determine whether there have been shifts in unintended drug exposure in this population (e.g., via adulterants) to inform prevention and harm reduction efforts. Methods: Adults entering nightclubs and festivals in New York City were asked about past-year drug use in 2016 through 2022, with a subset providing a hair sample for testing. We focused on the 1943 who reported ecstasy use (of which 247 had a hair sample analyzed) and compared trends in self-reported drug use, drug positivity, and adjusted prevalence (adjusting for unreported use). Results: MDMA positivity decreased from 74.4 % to 42.3 %, and decreases occurred regarding detection of synthetic cathinones ("bath salts"; a 100.0 % decrease), MDA (a 76.9 % decrease), amphetamine (an 81.3 % decrease), methamphetamine (a 64.2 % decrease), and ketamine (a 33.4 % decrease) (ps < .05). Although prevalence of MDA and synthetic cathinone use was comparable between self-report and adjusted report in 2022, gaps in prevalence were wider in 2016 (ps < .01). Adjusted prevalence of synthetic cathinone use decreased more across time than prevalence based on self-report (a 79.4 % vs. 69.1 % decrease) and adjusted report for MDA use decreased more than prevalence based on self-report (a 50.6 % vs. 38.9 % decrease). Conclusions: Combining self-report and toxicology tests helped us determine that decreases in drug use/exposure were steeper regarding adjusted prevalence. Underreported drug exposure-possibly due to exposure to adulterants-appears to have had less of an effect on prevalence in 2022 than it did in 2016.

Are the NPS commonly used? An extensive investigation in Northern Italy based on hair analysis.

New psychoactive substances (NPS) are present on the Italian illicit markets, but data from the analysis of biological samples to evaluate their real consumption are rare. For this reason, an epidemiological study was carried out by means of a ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) method for the determination of 115 NPS on the keratin matrix. A total of 847 hair samples were collected in 2020 and 2021 and analyzed. The sample donors were in the age range of 18-40 years, from both genders, and were tested either for driving relicensing or for drug withdrawal monitoring. The UPLC-MS-MS system consisted of a Waters ACQUITY UPLC® I-Class, coupled with a Waters XEVO TQ-XS triple quadrupole mass spectrometer. The method was developed and fully validated according to international guidelines. Limits of detection were set as the minimum criterion to identify positive samples. Overall, 56 samples resulted positive for ketamine, 35 for norketamine, 6 for fentanyl, 3 for norfentanyl, 3 for 4-ANPP, 3 for MDMB-4en-PINACA, 2 for N,N-DMT, 2 for 5-chloro AB-PINACA, 1 for α-PHP and 1 for methcathinone. NPS were detected in a small part of samples (8.4%), which seems in contrast with their apparent wide diffusion in Italy, yet it is congruent with similar investigations based on hair analysis. Future studies will be performed to expand the investigated population, especially in terms of age and origin.

Detection of fentanyl, synthetic opioids, and ketamine in hair specimens from purposive samples of American and Italian populations.

With the current crisis related to the diffusion of fentanyl and other novel opioids in several countries and populations, new and effective approaches are needed to better elucidate the phenomenon. In this context, hair testing offers a unique perspective in the investigation of drug consumption, producing useful information in terms of exposure to psychoactive substances. In this research, we applied targeted ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) analytical methods to detect novel synthetic and prescription opioids and other common controlled psychoactive drugs in the keratin matrix. A total of 120 hair samples were analyzed from the United States (US) and Italy, segmented when longer than 6 cm, and then analyzed. In the 60 samples (83 segments in total) analyzed from a purposive sample of data collected in the US, fentanyl was detected in 14 cases (16.9%), with no detection of nitazens or brorphine. We also detected fentanyl metabolites, despropionyl-p-fluorofentanyl, and prescription opioids. In the 60 samples collected in Italy (91 segments in total), ketamine was the most prevalent compound detected (in 41 cases; 45.1%), with ketamine demonstrating a strong correlation with detection of amphetamines and MDMA, likely due to co-use of these substances in recreational contexts. Several common drugs were also detected but no exposure to fentanyl or its analogs were detected. Results of this retrospective exploration of drug use add to increasing evidence that hair testing can serve as a useful adjunct to epidemiology studies that seek to determine biologically confirmed use and exposure in high-risk populations.

Oxidative Stress in a Mother Consuming Alcohol during Pregnancy and in Her Newborn: A Case Report.

Fetal alcohol spectrum disorder (FASD) is a set of conditions resulting from prenatal alcohol exposure (PAE). FASD is estimated to affect between 2% and 5% of people in the United States and Western Europe. The exact teratogenic mechanism of alcohol on fetal development is still unclear. Ethanol (EtOH) contributes to the malfunctioning of the neurological system in children exposed in utero by decreasing glutathione peroxidase action, with an increase in the production of reactive oxygen species (ROS), which causes oxidative stress. We report a case of a mother with declared alcohol abuse and cigarette smoking during pregnancy. By analyzing the ethyl glucuronide (EtG, a metabolite of alcohol) and the nicotine/cotinine in the mother's hair and meconium, we confirmed the alcohol and smoking abuse magnitude. We also found that the mother during pregnancy was a cocaine abuser. As a result, her newborn was diagnosed with fetal alcohol syndrome (FAS). At the time of the delivery, the mother, but not the newborn, had an elevation in oxidative stress. However, the infant, a few days later, displayed marked potentiation in oxidative stress. The clinical complexity of the events involving the infant was presented and discussed, underlining also the importance that for cases of FASD, it is crucial to have more intensive hospital monitoring and controls during the initial days.

Wastewater surveillance of 105 pharmaceutical drugs and metabolites by means of ultra-high-performance liquid-chromatography-tandem high resolution mass spectrometry.

Water pollution from pharmaceutical drugs is becoming an environmental issue of increasing concern, making water quality monitoring a crucial priority to safeguard public health. In particular, the presence of antidepressants, benzodiazepines, antiepileptics, and antipsychotics require specific attention as they are known to be harmful to aquatic biota. In this study, a multi-class comprehensive method for the detection of 105 pharmaceutical residues in small (30 mL) water samples was developed according to fit-for-purpose criteria and then applied to provide wide screening of samples obtained from four Wastewater Treatment Plants (WWTPs) in northern Italy. The filtered samples (0.22 µm filters) were extracted by SPE, and then eluted. 5 µL of the concentrated samples were analyzed by a UHPLC-QTOF-HRMS method validated for screening purposes. Adequate sensitivity was recorded for all target analytes, with limits of detection below 5 ng/L for 76 out of 105 analytes. A total of 23 out of the 105 targeted pharmaceutical drugs was detected in all samples. Several further compounds were detected over wide concentration intervals, ranging from ng/L to µg/L. In addition, the retrospective analysis of full-scan QTOF-HRMS data was exploited to carry out an untargeted screening of some drugs' metabolites. As a proof of concept, it was investigated the presence of the carbamazepine metabolites, which is among the most frequently detected contaminants of emerging concern in wastewater. Thanks to this approach, 10,11-dihydro-10-hydroxycarbamazepine, 10,11-dihydro-10,11-dihydroxycarbamazepine and carbamazepine-10,11-epoxide were identified, the latter requiring particular attention, since it exhibits antiepileptic properties similar to carbamazepine and potential neurotoxic effects in living organism.

Metabolic study of new psychoactive substance methoxpropamine in mice by UHPLC-QTOF-HRMS.

Methoxpropamine (MXPr) is an arylcyclohexylamine dissociative drug structurally similar to 3-methoxyeticyclidine, ketamine, and deschloroketamine, recently appeared in the European illegal market, and was classified within the new psychoactive substances (NPS). Our study investigated the metabolism of MXPr to elucidate the distribution of the parent drug and its metabolites in body fluids and fur of 16 mice. After the intraperitoneal administration of MXPr (1, 3, and 10 mg/kg), urine samples from eight male and eight female mice were collected every hour for six consecutive hours and then at 12- to 24-h intervals. Additionally, plasma samples were collected 24 h after MXPr (1 and 3 mg/kg) administration. Urine and plasma were diluted 1:3 with acetonitrile/methanol (95:5) and directly injected into the UHPLC-QTOF-HRMS system. The phase-I and phase-II metabolites were preliminarily identified by means of the fragmentation patterns and the exact masses of both their precursor and fragment ions. Lastly, the mice fur was analyzed following an extraction procedure specific for the keratin matrix. Desmethyl-MXPr-glucoronide was identified in urine as the main metabolite, detected up to 24 h after administration. The presence of norMXPr in urine, plasma, and fur was also relevant, following a N-dealkylation process of the parent drug. Other metabolites that were identified in fur and plasma included desmethyl-MXPr and dihydro-MXPr. Knowledge of the MXPr metabolites evolution is likely to support their introduction as target compounds in NPS toxicological screening analysis on real samples, both to confirm intake and extend the detection window of the dissociative drug MXPr in the biological matrices.

Recent advances in the detection of drugs of abuse by dried blood spots.

The purpose of this review study was to sum up the main recent advances reported in the scientific literature about the detection of common drugs of abuse in biological samples upon their collection by dried blood spot devices. The most recent, innovative and fully validated methods for the qualitative and/or quantitative detection of common drugs of abuse are reported, including alprazolam, clonazepam, diazepam, 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methyl-enedioxyethylamphetamine, amphetamine, methamphetamine, cocaine, tetrahydrocannabinol, 6-monoacetylmorphine, morphine, codeine, hydromorphone, hydrocodone, oxycodone, noroxycodone, tramadol, methadone, buprenorphine, fentanyl, ketamine and their respective metabolites and γ-hydroxybutyric acid. Dried blood spot proved to be extremely promising for routine analysis of forensic cases, although large-scale experiments on real samples need to be performed to confirm the emerging advantages of the technique and remove the potential limitations still affecting its widespread application.

Sexual Harassments Related to Alcohol and Drugs Intake: The Experience of the Rape Centre of Turin.

A cross-sectional study was conducted that describes the characteristics of sexual violence episodes related to the intake of alcohol and drugs observed among women that turned to the "Centro Soccorso Violenza Sessuale" (SVS) of the Sant'Anna Hospital in Turin between 1 January 2008, and 31 December 2017. Two hundred twenty-two patients were enrolled, 25 of which were minors, 141 were Italians, and most of them knew their aggressor and were raped in a private home. One hundred and fifty-five of them declared to the healthcare personnel to have taken alcoholic substances and/or drugs in conjunction with the event (86 reported having drunk alcohol, 36 having taken drugs and 33 disclosed both alcohol and drug abuse). If the woman knew her abuser, alcohol consumption was described as voluntary in more than 80% of cases, while in relation to drugs the consumption was equally voluntary or fraudulent. About 73% of women who reported having drunk alcohol just had amnesia or amnesia related to other symptoms, while amnesia was present in about 63% of women who reported only drug use. Physicians observed physical injuries on 156 women. Patients who reported to have assumed alcohol presented a significantly higher risk to suffer any physical injury and have a significantly increased risk to suffer injuries to their head and/or neck. The results obtained underline how even in Northern Italy alcohol intake represents the most widespread psychoactive substance in case of drug-facilitated sexual assault. There is therefore a need to promote education and prevention campaigns among citizens, especially among the youngest.

GC-MS Identification and Quantification of the Synthetic Cannabinoid MDMB-4en- PINACA in Cannabis-derived Material Seized in the Turin Metropolitan Area (Italy).

BACKGROUND: The presence of the synthetic cannabinoid receptor agonist MDMB-4en-PINACA in adulterated low-THC cannabis products was recently highlighted in several reports. Moreover, numerous intoxication cases involving MDMB-4en-PINACA have been described. OBJECTIVE: In order to monitor the diffusion of cannabis products containing MDMB-4en-PINACA in our territory, a total of 358 cannabis-derived samples (213 vegetal material and 145 resins) seized in the period November 2020 - February 2021 in the western Piedmont Area (Italy) was analyzed. METHODS: General screening analyses for traditional and synthetic cannabinoids were performed by a GC-MS device operating in full scan mode (40-600 amu). The MDMB-4en-PINACA was quantified by means of a specific GC-SIM-MS protocol purposely developed and validated, while the quantification of THC, CBD, and CBN was carried out by a GC-SIM-MS method routinely employed in our laboratory. RESULTS: MDMB-4en-PINACA was detected in 12 out of 358 samples (3.4% of the total). Among these, the molecule was found in 11 vegetal materials and in one resin sample. Considering solely the analysis of the 213 herb products, a positive rate of 5.2% was found for the presence of MDMB-4en-PINACA in these samples. MDMB-4en-PINACA was found in the seized materials at concentration levels ranging from 0.4 up to 6.3 mg/g (mean 2.5 mg/g; median 1.7 mg/g). Concerning the traditional cannabinoids, the THC concentration was in the interval 3-43 mg/g (mean 12 mg/g; median 7 mg/g), while CBD was found at higher concentrations in all specimens, specifically in the range 47-140 mg/g (mean 87 mg/g; median 80 mg/g). CONCLUSION: The adulteration of low-THC cannabis products with synthetic cannabinoid receptor agonists is widespread today. Since these substances are potentially more toxic than THC, their consumption poses a high risk of overdose for unaware users and a health-threatening situation. This study confirmed the sporadic presence on the market of CBD-prevalent cannabis products adulterated with MDMB-4en-PINACA.

Development and Validation of a GC-EI-MS/MS Method for Ethyl Glucuronide Quantification in Human Hair.

Ethyl glucuronide (EtG) is a minor, non-oxidative ethanol metabolite detectable in several matrices for specific periods of time. In recent years, quantification of EtG in hair has been established as the most reliable biomarker for long-term alcohol consumption, with the Society of Hair Testing offering cut-off values for assessment of both abstinence and heavy drinking. Instrumental constrains and wide inter- and intra-laboratory variability represent the ultimate barriers to widespread acceptance of hair EtG determination in the forensic context. In this study, a new analytical method for hair EtG based on gas chromatographic (GC) separation, electron impact (EI) ionization, and tandem mass spectrometry (MS/MS) detection was developed and validated. At the same time, several parameters for sample pretreatment and instrumental analysis were optimized using real hair samples obtained from different drinking subjects. A full-factorial design-of-experiment approach included procedures for hair washing, pulverization, and extraction. Rigorous multi-step washing proved not to reduce the EtG content extracted in the subsequent sample incubation. Hair pulverization with a ball mill significantly improved the EtG extraction from the keratin matrix and allowed us to reduce the time needed for the subsequent extraction step, without affecting the extraction recovery. The hair extract was derivatized with N-methyl-N-(trimethylsilyl)-trifluoroacetamide. Upon electron impact ionization of the EtG-TMS derivative, triple quadrupole mass analyzers were operated in the selected reaction monitoring (SRM) mode using the fragment m/z 405 as the precursor ion (m/z 410 for the EtG-D5 internal standard), the transitions m/z 405 → 359 and m/z 410 → 359 for quantitation, and m/z 405 → 331 and m/z 405 → 287 for qualification/confirmation, all at 10 V collision energy. The final method was fully validated and then applied to 25 real hair samples. The calibration curve proved linear between 6 and 60 pg/mg. The limit of detection (LOD) was 4 pg/mg. Intra- and inter-assay precision and accuracy tests showed a variability and bias close to 15% or lower over the entire calibration range. The new method is routinely applied in the Italian State Police's toxicology laboratory for hair analyses addressed to exclude excessive alcohol drinking and verify the psycho-physical requirements of the personnel.

Development and validation of a UHPLC-HRMS-QTOF method for the detection of 132 New Psychoactive Substances and synthetic opioids, including fentanyl, in Dried Blood Spots.

Dried Blood Spots (DBS) represents a promising micro-sampling technique in the field of forensic toxicology to carry out minimally invasive blood sample collection. In DBS, cheap, fast and easy sampling is combined with effortless store and transport. These properties aimed us to develop and validate a quick and easy procedure for the detection of a large and diverse range of emerging and alarming New Psychoactive Substances (NPS). A drop of whole blood sample was collected on a DBS card and dried for 3 h, from which a total of 132 analytes (including NPS, synthetic opioids NSO and metabolites) plus 13 deuterated internal standards could be extracted using 500 µL of a methanol/acetonitrile mixture (3:1, v/v) and subsequently separated and identified by means of ultra-high-performance liquid-chromatography (UHPLC) coupled to high resolution mass spectrometry (HRMS). The extraction efficiency proved to be reproducible with yields ranging from 30% to 100% depending on the different classes of drugs. Trueness, repeatability, and intermediate precision fulfilled acceptance criteria for almost all synthetic opioids, cathinones and hallucinogens (bias and CV% below ±20%); in particular, the aggregate inter-day trueness data showed extremely limited deviation from the expected concentrations (-10% < bias% < +10%) for 114 target analytes out of 132. The calculated limits of detection ranged from 1.3 to 6.3 ng/mL, consistently exceeding the values experimentally tested. Moderate ion suppression was observed for most analytes, partly caused by blood fortification itself. Good stability of the target analytes at -20 °C, 4 °C, and 35 °C on DBS cards after drying was observed, even for long periods of time. Optimal storage condition appeared to be at 4 °C resulting in virtually no drugs degradation for up to 40 days. The novel analytical method based on DBS sampling, verified on venous whole blood real samples previously tested positive with our routine procedure, conveys remarkable potential in analytical toxicology, clinical analysis, and doping control.