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.

"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.

A green analytical method for the simultaneous determination of 17 perfluoroalkyl substances (PFAS) in human serum and semen by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS).

The ubiquity of perfluoroalkyl substances has raised concerns about the unintended consequences of PFAS exposure on human health. In the present study, an eco-friendly ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed for the simultaneous determination of 17 PFAS in human serum and semen samples. QuEChERS salts MgSO4:NaCl 4:1 (w/w) were used for the extraction. The separation of analytes was performed on an ACQUITY BEH C18 column (100 × 2.1 mm, 1.7 µm), using water:methanol 95:5 and methanol as mobile phases A and B, respectively, both containing 2 mM ammonium acetate. Multiple reaction monitoring (MRM) in negative ion mode was used, selecting two transitions for each analyte, except for perfluorobutanoic acid (PFBA) and perfluoropentanoic acid (PFPeA). The analytical method was validated according to the Organization of Scientific Area Committees (OSAC) for Forensic Sciences guidelines and AGREE approach software was used to evaluate the greenness of the method. The developed procedure was applied to the analysis of 10 paired human serum and semen samples, proving the suitability in high throughput laboratories due to the easy preparation and the reduced volume of toxic solvents. Moreover, it allows to perform further investigation on the correlation between serum and semen PFAS concentration, focusing on male reproductive system correlated pathologies, such as male infertility.

NPS-EQA PART II: Four years' experience in external quality assessment program in Italy for classical and new psychoactive substances analysis in hair.

In 2019, the Italian National Institute of Health established an external quality assessment (EQA) program to evaluate the performance of laboratories of collaborative centres participating in the National Early Warning System in hair testing for classical and new psychoactive substances (NPS). The results obtained in the four rounds (2019-2023) and the evolution in hair testing performance for classic drugs of abuse and new psychoactive substances are presented. A total of 11 hair specimens, including 3 blank samples, were prepared by adding different classes of classical and NPS at known concentrations to pre-screened drug-free hair. False negative and false positive results were calculated for the qualitative data evaluation. The quantitative evaluation included the imprecision (as % coefficient of variation, CV%) and the accuracy (as % error, ERR%) of the results with respect to a mean value obtained by reference laboratories and Z-score values were assessed. Over the years, an improvement in false negative results (from 52.4% in the first year to 34.3% in the last one) and false positive results (from 55.0% in the first year to 30.8.% in the last one) was observed. In the first round, the mean ERR% ranged from 6.2% to 112.8% due to NPS determination. However, in the subsequent three rounds, the mean ERR% ranged from 10.4% to 22.4%, The mean CV% in the four rounds was approximately 41.5% (ranging from 44.3% to 53.3%). Between 12.0% and 56.6% of the reported results in all rounds should be considered satisfactory. EQA programs help laboratories to identify and correct problems within their processes by highlighting errors and variations. This ensures that the results produced are accurate and reproducible.

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.

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.

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.

Usefulness of Oral Fluid for Measurement of Methylone and Its Metabolites: Correlation with Plasma Drug Concentrations and the Effect of Oral Fluid pH.

The aim of this study was to investigate methylone and its metabolites concentration in oral fluid following controlled increasing doses, focusing on the effect of oral fluid pH. Samples were obtained from a clinical trial where twelve healthy volunteers participated after ingestion of 50, 100, 150 and 200 mg of methylone. Concentration of methylone and its metabolites 4-hydroxy-3-methoxy-N-methylcathinone (HMMC) and 3,4-methylenedioxycathinone in oral fluid were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Pharmacokinetic parameters were estimated, and the oral fluid-to-plasma ratio (OF/P) at each time interval was calculated and correlated with the oral fluid pH using data from our previous study in plasma. Methylone was detected at all time intervals after each dose; MDC and HMMC were not detectable after the lowest dose. Oral fluid concentrations of methylone ranged between 88.3-503.8, 85.5-5002.3, 182.8-13,201.8 and 214.6-22,684.6 ng/mL following 50, 100, 150 and 200 mg doses, respectively, peaked between 1.5 and 2.0 h, and were followed by a progressive decrease. Oral fluid pH was demonstrated to be affected by methylone administration. Oral fluid is a valid alternative to plasma for methylone determination for clinical and toxicological studies, allowing for a simple, easy and non-invasive sample collection.

Recent challenges and trends in forensic analysis: Δ9-THC isomers pharmacology, toxicology and analysis.

Δ9-tetrahydrocannabinol (Δ9-THC) isomers, especially Δ8-tetrahydrocannabinol (Δ8-THC), are increasing in foods, beverages, and e-cigarettes liquids. A major factor is passage of the Agriculture Improvement Act (AIA) that removed hemp containing less than 0.3 % Δ9-THC from the definition of "marijuana" or cannabis. CBD-rich hemp flooded the market resulting in excess product that could be subjected to CBD cyclization to produce Δ8-THC. This process utilizes strong acid and yields toxic byproducts that frequently are not removed prior to sale and are currently inadequately studied. Pharmacological activity is qualitatively similar for Δ8-THC and Δ9-THC, but most preclinical studies in mice, rats, and monkeys documented greater ∆9-THC potency. Both isomers caused graded dose-response effects on euphoria, blurred vision, mental confusion and lethargy, although Δ8-THC was at least 25 % less potent. The most common analytical methodologies providing baseline resolution of ∆8-THC and ∆9-THC in non-biological matrices are liquid-chromatography coupled to diode-array detection (LC-DAD or LC-PDA), while liquid chromatography coupled to mass spectrometry is preferred for biological matrices. Other available analytical methods are gas-chromatography-mass spectrometry (GC-MS) and quantitative nuclear magnetic resonance (QNMR). Current knowledge on the pharmacology of ∆8-THC and other ∆9-THC isomers are reviewed to raise awareness of the activity of these isomers in cannabis products, as well as analytical methods to discriminate ∆9-THC qualitatively, and quantitatively and ∆8-THC in biological and non-biological matrices.