An LC-MS/MS method for the determination of drugs of abuse included THC-COOH, EtG, and NPS, using a single hair extraction sample.

Hair analysis plays an important role in the determination of drugs of abuse in both forensic and clinical toxicology investigations. The analysis of different substances often requires the use of different sample preparation methods, thereby increasing the amount of hair sample and time required. In the present study, a fast method involving a combination of a single 25 mg hair extraction procedure and four liquid chromatography-tandem mass spectrometry methods using the same chromatographic phases and column was developed and validated. The target was the identification and quantification of various commonly abused drugs and their metabolites, including amphetamines, cocaine, opioids, cannabinoids, THC-COOH and EtG, and more than 140 new psychoactive substances, including synthetic cannabinoids, phenethylamines, synthetic opioids, methylphenidate, cathinone, piperidine, and tryptamines.

Sex differences and driving impairment related to psychoactive substances.

OBJECTIVE: The first aim of the study was to identify sex differences in the use of psychoactive substances among subjects with a previous driving under the influence (DUI) episode. The secondary objective was to propose specific strategies for medico-legal improvements. METHODS: This was a retrospective observational study that took place between June 1, 2019, and August 31, 2023. It was conducted on DUI subjects examined for reinstatement of their driver's license using an integrated medico-legal and toxicological approach. Ethyl glucuronide (EtG) and illicit psychoactive substances were determined from hair samples. We performed descriptive statistical analyses for the entire sample as well as separately by sex. Additionally, we conducted binary logistic regression analyses separately for males and females to identify protective/risk factors associated with previous road accidents and judgments of unfitness to drive due to excessive alcohol consumption (EtG ≥ 30 pg/mg). RESULTS: The study included 2,221 subjects, comprising 1,970 men and 251 women. Men exhibited a higher prevalence of tobacco, alcohol, and illicit psychoactive substance use. Women were more frequently co-users of alcohol and psychoactive substances and involved in road accidents at the time of DUI. Among the men, being married or having a partner was found to be a protective factor concerning past traffic accidents. For both sexes, a DUI episode with a blood alcohol concentration (BAC) exceeding 1.5 g/L or the co-ingestion of alcohol and drugs was identified as a risk factor for road accident involvement. For men, smoking more than 20 cigarettes per day and, for women, having a DUI episode with a BAC over 1.5 g/L were the main factors indicating unfitness to drive, as determined through high hair EtG levels (> 30 pg/mg). Women with a previous history of road accidents were less likely to have EtG levels of 30 pg/mg or more. CONCLUSIONS: The study confirmed sex differences in subjects with a previous DUI episode. A BAC exceeding 1.5 g/L or the simultaneous use of alcohol and drugs at the time of DUI necessitate careful assessment of both men and women seeking driver's license reinstatement. In women, a BAC exceeding 1.5 g/L is considered a risk factor for a subsequent judgment of unfitness to drive. The medico-legal assessment should also involve a thorough investigation of smoking habits in men, as these habits could be related to an increased risk of excessive alcohol consumption.

Hair toxicological analysis of infants and their mothers: a 5-year retrospective study focusing on cocaine.

Prenatal and infant exposure to drugs of abuse is an emerging social and public health problem affecting children health and which may relate to child abuse and neglect. Exposure to drugs of abuse may occur through different routes, including intrauterine, breastfeeding, accidental intake, passive inhalation, and intentional administration. Currently, cases of suspected exposure can be investigated by hair toxicological analysis, the interpretation of which is, however, often difficult, leading to consequent difficulties in the management of such cases. In order to provide a contribution in terms of interpretation of the analytical results, this study aimed to search for the possible existence of elements, from a toxicological point of view, indicative towards the route of exposure. A retrospective study was performed on cases of suspected exposure to drugs of abuse in children aged 0-1 year, evaluated at a University Hospital between 2018 and 2022. Data of children hair toxicological analysis were analyzed and then compared with those of their mothers, when available; 41.6% children tested positive for cocaine. The study found a significant correlation between cocaine and benzoylecgonine concentrations, and a benzoylecgonine/cocaine ratio that tends to decrease as the age of children increases. From the comparison with mothers, a child/mother cocaine concentration ratio lower than 1 was found in all cases of hair sampled within the first week of life, and a ratio greater than or equal to 1 in all cases in which the sampling was performed later. These results, if confirmed in a larger cohort, could represent a contribution in the interpretation of cases of infant exposure to drugs of abuse and be integrated in the context of their multidisciplinary evaluation.

Comparison of two methods for the extraction of ethylglucuronide from hair.

The aim was the comparison between the Society of Hair Testing (SoHT) consensus for the use of alcohol markers which powdering hair for the extraction of ethylglucuronide (EtG) in water and extraction using the patented M3 Reagent Test kit on cut hair. Hair samples were cut into small segments and washed twice with methanol and diethyl ether. The SoHT-Consensus entails the extraction of pulverised hair in water. This is obtained by incubation of 25 mg of hair at room temperature overnight and 2 h sonication, even if the overnight incubation is not mandatory. The M3 method entails incubation of 25 mg of cut hair with the M3-Reagent at 100°C for 60 min. After centrifugation, the supernatant is injected into a liquid chromatography-tandem mass spectrometry (LC-MS/MS). Samples (191) were collected in the APSS laboratory in Trento, Italy, between 2021 and 2022. The limit of quantification (LOQ) was set at 5 pg/mg for the pulverised and M3-Reagent methods. Assays showed good linearity above the range of LOQ-300 pg/mg. Precision (within 20%) values were also obtained using both methods. In the Passing-Bablock linear regression, the final regression curve between M3 (y) and the pulverising method (x) showed good agreement; the Bland-Altman analysis did not show any significant bias between the two methods. The M3-Reagent method, due to cut hair use, is easy to perform, saves time and allows for a smaller sample quantity loss with use of nondisposable grinding jars for the ball mill to obtain the extraction of EtG.

Driving under the influence of cannabis: A 5-year retrospective Italian study.

INTRODUCTION: Cannabis consumption is associated with driving impairment and increased crash risk, endangering road safety. Toxicological analyses play a fundamental role in detecting a recent consumption of psychoactive substances. The aim of this study was to examine the concentration of cannabinoids in blood samples of driving-under-the-influence (DUI) offenders in order to investigate whether delayed sample collection affects the toxicological assessment of the offenders. MATERIALS AND METHODS: An observational retrospective study was performed using anonymized toxicological data referring to cannabis-related DUI offenders involved in road traffic accidents (RTA) or apprehended by the police from 1 January 2017-31 December 2021 archived at Legal Medicine and Toxicology Department of the University Hospital of Padova, Italy. RESULTS: In a total sample of 318 drivers, 143 blood samples tested positive for tetrahydrocannabinol (THC) and metabolites 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC) and 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH), and 173 blood samples were positive for THC-COOH with THC negative. In the first group, the mean concentrations of THC and THC-COOH were 4.05 ng/mL and 28.29 ng/mL, respectively. In THC-negative cases, the mean THC-COOH concentration was 7.3 ng/mL. The time elapsed between the event and sample collection varied from 15 min to 7 h (mean 2 h 29 min). The average estimated time elapsed after consumption of cannabinoids was 3 h 7 min (Model I) and 2 h 36 min (Model II). CONCLUSIONS: The present research discussed the main difficulties in the toxicological evaluation of drivers under the influence of Cannabis. Issues related to the time between RTA and sample collection, the laws and legal limits in force in various Countries were presented.

Characterization of URB Series Synthetic Cannabinoids by HRMS and UHPLC-MS/MS.

A large number of synthetic cannabinoids are included in new psychoactive substances (NPS) and constitute an open research area in analytical pharmaceutical and toxicology when methods are needed to unambiguously identify these substances and their metabolites in biological fluids. A full molecular characterization of five synthetic molecules of the URB series that is able to interact with the endocannabinoid system was achieved with a high-resolution mass spectrometry (HRMS) in positive ion electrospray ionization and collisional experiments on the protonated parent ions, obtaining characteristic fragmentation patterns. Ultra-high-performance liquid chromatography coupled with a triple quadrupole (UHPLC-MS/MS) has also been used, which can help develop methods for screening and confirming synthetic cannabinoids in biological fluids.

Assessing maternal alcohol consumption in pregnancy: comparison of confidential postnatal maternal interview and measurement of alcohol biomarkers in meconium.

OBJECTIVE: Knowledge of alcohol consumption in pregnancy is important for early identification of children with fetal alcohol spectrum disorder. We investigated whether alcohol biomarkers fatty acid ethyl esters (FAEEs) and ethyl glucuronide (EtG) in meconium are predicted by maternal or newborn demographics and/or correlate with confidential early postnatal self-report of alcohol consumption in pregnancy. DESIGN: Anonymised, observational population-based study. SETTING: Inner-city maternity unit, Glasgow, UK. PATIENTS: Singleton mother/infant dyads delivering every fourth day. INTERVENTIONS: Mother: confidential postnatal interview. Baby: meconium sample for FAEEs and EtG. RESULTS: 840/908 mothers consented. 370 (46.4%) reported alcohol consumption in pregnancy, generally of modest amount; for 114 (13.6%) this was after 20 weeks' gestation. Alcohol consumption in later pregnancy was more commonly reported by older (31.3 vs 29.5 years) women of white British ethnicity (p<0.05); their babies were on average 118 g heavier (p=0.032). FAEEs were identified in all meconium samples; concentration was ≥600 ng/g in 39.6%. EtG concentration was ≥30 ng/g in 14.5%. Neither biomarker was associated with maternal age, body mass index or socioeconomic status but when EtG was ≥30 ng/g, the mother was less likely to identify as white British (71.3% vs 81.8%, p=0.028). Sensitivities of FAEEs ≥600 ng/g and EtG ≥30 ng/g were 43.1% and 11.6%, respectively for postnatal self-report of alcohol use in later pregnancy (specificities 60.6% and 84.8%). CONCLUSIONS: FAEEs and EtG measured in meconium have low sensitivity and specificity for self-reported alcohol consumption after 20 weeks' gestation in an unselected Scottish population.

Assessing maternal alcohol consumption in pregnancy: does phosphatidylethanol measured from day 5 newborn blood spot cards have any value? An observational, population-based study.

OBJECTIVE: Prenatal alcohol exposure (PAE) places children at risk of fetal alcohol spectrum disorder (FASD) but ascertainment of PAE is problematic. Early intervention for children at risk of FASD may help mitigate long-term difficulties. Phosphatidylethanol (PEth), a metabolite of alcohol, is incorporated into red cell membranes and can be measured in dried blood spot (DBS) cards. In the UK, DBS samples are collected on day 5 for routine newborn screening. We sought to examine if PEth measured from DBS correlates with postnatal maternal self-report of alcohol consumption in pregnancy. DESIGN: Observational population-based study. Comparison of infant PEth concentration and self-report of maternal alcohol use during pregnancy. SETTING: Large maternity unit in Glasgow, Scotland. PARTICIPANTS: All singleton mother-infant dyads delivered during each fourth consecutive 24-hour period. INTERVENTIONS: Mother: direct, confidential, immediate postnatal interview by a single researcher examining alcohol use during pregnancy. Infant: one extra DBS collected coincident with routine newborn screening if bleeding continued. RESULTS: 92.5% of eligible mothers agreed to participate. 510 DBS were obtained of which 502 were successfully analysed. 216 (43%) samples contained PEth at a concentration of ≥8 ng/mL and 148 (29.5%) at ≥20 ng/mL. The sensitivity of PEth ≥8 ng/mL and ≥20 ng/mL in identifying women who self-reported modest alcohol use after 36 weeks' gestation was 50% and 36.4%, respectively. CONCLUSION: PEth measured from DBS obtained on day 5 of life does not reliably identify modest PAE after 36 weeks' gestation from maternal self-report.

Driving license regranting: Hair EtG, serum CDT, and the role of sociodemographic and medicolegal variables.

Driving under the influence (DUI) of alcohol is a road safety problem. Driving license regranting is based on the evaluation of medicolegal and toxicological variables that may include serum carbohydrate-deficient transferrin (CDT) and hair ethyl glucuronide (hEtG). The aim of the study was to compare the diagnostic performance of CDT and hEtG in a population of DUI offenders. Other factors potentially associated with heavy alcohol use were explored. The population included DUI offenders examined during the period of January 1, 2019, through June 30, 2022. Sociodemographic, medicolegal, and toxicological variables were collected. CDT in serum and EtG in head hair were determined in all subjects. Excessive alcohol intake (hEtG ≥30 pg/mg) was considered cause for unfitness to drive. Cohen's kappa coefficient was calculated. Descriptive analyses were performed using chi-square and Mann-Whitney tests. Variables significantly different between the groups were included in a multivariate binary logistic regression model. The sample encompassed 838 subjects (case group: 179, comparison group: 689). CDT exhibited poor agreement (κ = 0.053) with hEtG as the reference test. Lower education, age at DUI, heavy smoking, and GGT levels associated with heavy alcohol consumption differentiated the two groups. For DUI offenders, the use of CDT to assess heavy alcohol consumption is limited, possibly due to the time-window assessed, the time required for normalization, and the different amount of ethanol needed to reach higher CDT levels, in comparison to hEtG; thus, hEtG assessment is strongly recommended for this population. Heavy smoking, GGT, education, and age could be related to heavy alcohol consumption and higher risk of DUI.

Post-mortem investigation into a death involving doping agents: The case of a body builder.

INTRODUCTION: A young male was found dead on the bed of a hotel room. He was expected to take part in a bodybuilding competition the day after. During the site inspection, drugs of different types were found. The next day, an autopsy was performed. The evidence of cardiomegaly with organ congestion involving lung, liver, kidneys, adrenal glands, spleen and brain was confirmed by both the autoptic and the histopathological exam. However, the cause of death needed to be investigated. METHODS: A thorough toxicological investigation was undertaken by gas chromatography-mass spectrometry (GC-MS), liquid chromatography-high resolution mass spectrometry (LC-HRMS) and liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) on samples of urine, blood and hair. RESULTS AND DISCUSSION: Clenbuterol, a long-acting selective beta2 agonist, was found in both blood (1 ng/ml) and urine (1 ng/ml), and evidence of its use was provided by the analysis of the 3-cm hair (25 pg/mg). The main metabolite of drostanolone (2 alpha-methyl-androsterone), an anabolic steroid, was found in the urine (202 ng/ml), where an increased ratio of testosterone/epitestosterone (T/E = 11) emerged. Due to the results of the hair analysis, a long-term use of various anabolic steroids was supposed. The integrated analysis of the results and the absence of other possible causes (such as trauma or cardiac conduction anomalies) led to the identification of the abuse of doping substances as the underlying cause of death. CONCLUSION: Hair analysis has proven to be crucial in identifying drug misuse and the contributing cause of death.

Is Cannabis Legalization Eliciting Abusive Behaviors in Parents? A Case Report.

The interest in cannabis, cannabis-based compounds, and treatments is rapidly growing along with the legalization of marijuana in many countries and widespread use of cannabis derivatives in medical products. A growing body of literature is warning about possible unintentional intoxication in children because of unregulated and unsupervised use of cannabinoids by parents; to our knowledge, very rarely have parental self-prescription and self-administration to their children (affected by neurologic or other disorders or no disorders at all) been reported. We report a 4-year-old child, suffering from an anti-N-methyl-D-aspartate receptor encephalitis, who was found unpredictably positive for cannabis and other illicit substances after drug testing was performed in order to investigate the child's treatment-resistant behavioral disturbances. Toxicologic analyses were also extended to the child's parents, who finally disclosed that they had deliberately administered a cannabis-derived product (cannabidiol extract) as a home remedy for managing their child's behavior. Careless with regard to the possible adverse effects and certain that the product was legal, they presumed there was no need for them to inform the physicians in charge of treating the child of this practice.

Prevalence and concentrations of sedative-hypnotic drugs in blood of drivers involved in road traffic crashes in the Padova region of Italy - not so easy to interpret.

BACKGROUND & OBJECTIVES: This study reports the prevalence and concentrations of sedative-hypnotic drugs as exemplified by benzodiazepines (BZD) and zolpidem (Z-hypnotic) in blood samples from drivers involved in road traffic accidents (RTA) in the Padova region of Italy. Another aim of the study was to estimate the prevalence of these drugs with concentrations in blood above the therapeutic intervals and above specific per se limits. METHODS: A total of 4066 blood samples collected from drivers involved in RTA were analysed for the presence of alcohol, drugs of abuse and medicinal drugs with sedative-hypnotic properties. Prevalence of drivers positive for BZDs and zolpidem were reported according to the reporting limit of our laboratory (1 ng/mL) in a sort of zero tolerance approach and compared with the prevalence according to analytical cut-offs used in the "European Union's research project on Driving Under the Influence of Drugs, Alcohol and Medicines" (DRUID). The impairment-based, per se limits adopted in Norway and in England and Wales and the values used to define "therapeutic ranges" in blood and in plasma/serum were also applied to the case study. RESULTS: 175 blood samples were positive for sedative-hypnotics above 1 ng/mL, with the following prevalence: diazepam 44%, nordazepam 41.8%, lorazepam 32.6%, zolpidem 28%, oxazepam 25.6%, alprazolam 16%, delorazepam 11,6%, lormetazepam 11,6%, temazepam 11.6%, clonazepam 11.6%, triazolam 6.9%, N-desalkylflurazepam 4.6%, bromazepam 2.3%. When applying DRUID analytical cut-offs, the prevalence of BZDs and zolpidem sharply decreases. Applying the impairing cut-offs used in Norway, 56% of positive samples were above the limits equivalent to a BAC of 0.2 g/L, 39% above the limits corresponding to 0.5 g/L, and 23% above the cut-off corresponding to 1.2 g/L. Only 1% of the drivers had drug concentrations above the per se concentration limits adopted in England and Wales [26]. When comparing blood levels with therapeutic ranges in plasma, bromazepam, lormetazepam and delorazepam were often found above the highest limits. The adjustment of the concentrations with the plasma-to-blood ratios causes a significant increase of cases above the therapeutic ranges in plasma. CONCLUSIONS: Sedative-hypnotic drugs are medicinal substances frequently identified in drivers involved in RTA, commonly in concentrations associated with driving impairment. Besides the concentrations of drugs in blood, several factors have to be considered to conclude that a driver was impaired. The frequent association with alcohol, cocaine and other BZDs, confirms the abuse potential of these medications.

Overdose of Quetiapine-A Case Report with QT Prolongation.

Quetiapine is an atypical antipsychotic drug used to treat bipolar disorder, schizophrenia, and major depressive disorder. Although several studies describe the adverse effects of intoxication with Quetiapine, only a few report an extreme overdose without comedications that lead to a life threat. We present a case of a 75-year-old male who tried to attempt suicide by ingesting 28 g of Quetiapine. During the management in the emergency department, both serum and urine samples were collected, allowing a complete pharmacokinetic analysis to be conducted, from the admission to the discharge.

Coronavirus lockdown: Excessive alcohol consumption and illicit substance use in DUI subjects.

OBJECTIVE: This study investigates the consequences of the SarS-CoV-2 outbreak and of the resulting control measures on alcohol and illicit substance use in a high-risk population for substance-related disorders, utilizing an integrated medico-legal and toxicological approach. METHODS: The research was structured as a retrospective case-control study of subjects found to be driving under the influence (DUI) of alcohol and/or other psychoactive substances who were examined for driver's license regranting. Alcohol and/or drug use was assessed by comparing cases examined in the period from May to August 2020 (immediately after the lockdown in Italy) to control subjects examined in the same period in 2019. DUI subjects were examined by an integrated approach, descriptive analyses were conducted, and significance was determined by chi-square and Mann-Whitney tests. Variables linked to the pandemic outbreak and resulting lockdown were investigated as predictive factors in determining unfitness to drive. RESULTS: Cases (281) were significantly different from controls (261) concerning the judgment of unfitness to drive (p<.001) and had more subjects with chronic excessive alcohol use and/or illicit substance use. The two groups were rather homogeneous concerning the other variables, except for a difference in blood alcohol concentration (BAC) at the time of DUI (p = .027). No statistical association was found between the investigated variables linked to the lockdown and the judgment of unfitness to drive. CONCLUSIONS: Chronic excessive alcohol consumption and illicit substance use were more frequently observed in cases, which suggests a possible correlation between the pandemic/lockdown restrictions and an increase in psychoactive substance misuse. While these potentially correlative factors are discussed in this article, they require further study. If confirmed, the results should be considered in forensic and clinical settings.

Does Eumelanin Oxidation Play a Role on the Photostability of Ethyl Glucuronide in Hair Exposed to Simulated Solar Radiation?

The action of solar radiation on the concentration of the ethanol metabolite ethyl glucuronide (EtG) in 40 hair samples of nonabstinent subjects was investigated. Hair samples of different colors were analyzed before and after irradiation with artificial sunlight under a light dose corresponding to 3 months of sun exposure. After irradiation, an increase of EtG concentration was detected in 55% of the samples ranging from 5% to 141%. In 16 cases, a concentration reduction ranging from -2% to -74% was observed. The measure of the level of pyrrole-2,3,5-tricarboxylic acid (PTCA), a marker of eumelanin oxidation, demonstrated the largest increase in oxidation in light brown hair where the greatest degradation of EtG was observed after irradiation. However, the rise of PTCA in all hair tested was accompanied by increase in EtG concentration in 8/10 samples and by decrease in 2/10, suggesting no correlation between the two markers. To verify if hair structure was modified by light, scanning electron microscopy (SEM) analysis was performed on irradiated hair of different colors and compared with the dark samples. SEM revealed modification of hair structure in all samples showing partial shaft exfoliation and reduction of hair thickness under the treatment with solar radiation.

A standardized postmortem protocol to assess the real burden of sudden infant death syndrome.

Sudden unexpected infant death (SUID) is a major cause of death in infants < 1 year of age. Sudden infant death syndrome (SIDS) is a SUID still unexplained after post-mortem examination. In 2014, a protocol of post-mortem investigation was introduced to assess both the prevalence and the etiopathogenesis of SUID. Our aim was to compare SUID data before and after the application of a standardized autopsy protocol of investigation. In the time interval 2004-2018, SUID cases occurring in the Veneto Region, North-East Italy, were referred to our Core Lab. Since 2014, a complete autopsy was performed, including gross and histological study with toxicologic and molecular analysis carried out at the referral center. A total of 36 SUIDs (22 M, mean age 95.5 ± 80 days), 17 before (group A) and 19 after (group B) 2014, were collected. In group A, only 1 (6%) resulted as explained SUID, due to lymphocytic myocarditis and 16 (94%) were SIDS. In group B, 8 were SIDS (42%) and 11 (58%) explained SUID cases (p < 0.01), consisting of interstitial pneumonia and bronchiolitis in 9 and lymphocytic myocarditis in 2 cases. Molecular analysis was positive for viruses in 8 of them (73%). In conclusion, since the application of a standardized protocol of post-mortem investigation, inflammatory, mostly infective, cardio-pulmonary diseases have been identified as the most common cause of SUID, with SIDS falling from 94 to 42% of SUID. Efforts must be made to implement a uniform autopsy protocol to provide reliable epidemiological data on SIDS.