Photodegradation of cannabidiol (CBD) and Δ9-THC in cannabis plant material.

Δ9-THC, the psychotropic cannabinoid in Cannabis sativa L., for many years has been the focus of all the pharmacological attention as the main promising principle of the plant. Recently, however, cannabidiol (CBD) has brought a sudden change in the scenario, exponentially increasing the interest in pharmacology as the main non-psychotropic cannabinoid with potential therapeutic, cosmetical and clinical applications. Although the reactivity of CBD and Δ9-THC has been considered, little attention has been paid to the possible photodegradation of these cannabinoids in the vegetal matrix and the data available in the literature are, in some cases, contradictory. The aim of the present work is to provide a characterization of the photochemical behaviour of CBD and Δ9-THC in three cannabis chemotypes, namely I (Δ9-THC 2.50%w/w), II (CBD:Δ9-THC 5.82%w/w:3.19%w/w) and III (CBD 3.02%w/w).

Determination of ethyl glucuronide in hair and self-reported alcohol consumption in university students.

Young individuals constitute an intriguing population, as their drinking habits are notably shaped by their perception of their peers' alcohol consumption. Nonetheless, excessive alcohol intake can have detrimental effects on academic performance, interpersonal relationships, and the risk and severity of accidents. This study reported the first data involving students enrolled from three universities on a voluntary basis for alcohol consumption evaluation. Alcohol consumption was assessed through questionnaires and EtG quantification in hair (hEtG) carried out by liquid chromatography-mass spectrometry (LC-MS/MS) analysis after a solid-phase extraction (SPE) purification step. The results of our study demonstrated that 77.1% of samples tested negative for hEtG or displayed hEtG ≤ 5 pg/mg. Particularly, the student population was not characterized by samples with hEtG indicative of chronic excessive consumption (hEtG ≥ 30 pg/mg). No significant association was identified between biological sex, among the degree course/the year attended, nor in relation to BMI or smoking/coffee consumption. Among the obtained results, it was worth noting that the comparison of self-reporting abstinence from tobacco and coffee accounted for 65.3% and 16.7%, respectively, while only 2.8% of the total declared abstinence from alcohol. The current study has uncovered a significant level of interest among students in this analysis and its interpretation. This suggests that implementing public health promotion activities within a university setting could be beneficial.

New Synthetic Cathinones and Phenylethylamine Derivatives Analysis in Hair: A Review.

The analysis of psychoactive substances in hair is of great importance for both clinical and forensic toxicologists since it allows one to evaluate past and continuative exposure to xenobiotics. In particular, a new challenge is represented by new psychoactive substances: Among this new class of drugs of abuse, synthetic cathinone and phenethylamine derivatives are often detected in biological samples. Hence, there is a growing need to develop new analytical procedures or improve old ones in order to conduct evaluations of these emerging substances. This study is a systematic review of all the instrumental and experimental data available in the literature. A total of 32 articles were included in the review. Acidic solvents proved to be the most reliable solutions for extraction. Gas chromatography and liquid chromatography coupled to tandem mass spectrometric and high-resolution mass spectrometric systems represent the majority of the involved instrumental techniques. Sensitivity must be maintained at the pg/mg level to detect any occurrences up to occasional consumption. In total, 23 out of 32 articles reported real positive samples. The most frequently detected substance in hair was mephedrone, followed by butylone, methylone, MDPV, and α-pyrrolidinophenone-type substances.

Comparison of Two Immunoassay Screening Methods and a LC-MS/MS in Detecting Traditional and Designer Benzodiazepines in Urine.

Sensitive and specific immunoassay screening methods for the detection of benzodiazepines in urine represent an important prerequisite for routine analysis in clinical and forensic toxicology. Moreover, emerging designer benzodiazepines force labs to keep their methodologies updated, in order to evaluate the reliability of the immunochemical techniques. This study aimed at evaluating the sensitivity and specificity of two different immunoassay methods for the detection of benzodiazepines in urine, through a comparison with the results obtained by a newly developed liquid chromatographic tandem mass spectrometric (LC-MS/MS) procedure. A cohort of authentic urine samples (N = 501) were processed, before and after a hydrolysis procedure, through two immunoassays and an LC-MS/MS method. The LC-MS/MS target procedure was optimized for monitoring 25 different molecules, among traditional and designer benzodiazepines, including some metabolites. At least one of the monitored substances was detected in 100 out of the 501 samples. A good specificity was observed for the two immunoassays (>0.99), independently of the cut-offs and the sample hydrolysis. The new kit demonstrated a fairly higher sensitivity, always higher than 0.90; in particular, a high cross-reactivity of the new immunoassay was observed for samples that tested positive for lorazepam and 7-aminoclonazepam. The two immunoassays appeared adequate to monitor not only traditional benzodiazepines but also new designer ones.

Analysis of Cannabinoids and Metabolites in Dried Urine Spots (DUS).

Dried urine spots (DUS) represent a potential alternative sample storage for forensic toxicological analysis. The aim of the current study was to develop and validate a liquid chromatographic tandem mass spectrometric procedure for the detection and quantitative determination of cannabinoids and metabolites in DUS. A two-step extraction was performed on DUS and urine samples. An LC-MS/MS system was operated in multiple reaction monitoring and positive polarization mode. The method was checked for sensitivity, specificity, linearity, accuracy, precision, recovery, matrix effects and carryover. The method was applied to 70 urine samples collected from healthy volunteers and drug addicts undergoing withdrawal treatment. The method was successfully developed for DUS. LODs lower than 2.0 ng/mL were obtained for all the monitored substances. All the validation parameters fulfilled the acceptance criteria either for DUS or urine. Among the real samples, 45 cases provided positive results for at least one compound. A good quali-quantitative agreement was obtained between DUS and urine. A good stability of THC, THCCOOH and THCCOOH-gluc was observed after a 24 h storage, in contrast to previously published results. DUS seems to provide a good alternative storage condition for urine that should be checked for the presence of cannabinoids and metabolites.

Determination of Antidepressants and Antipsychotics in Dried Blood Spots (DBSs) Collected from Post-Mortem Samples and Evaluation of the Stability over a Three-Month Period.

An LC-MS/MS method for the identification and quantification of antidepressants and antipsychotics was developed on dried blood spots (DBSs). Moreover, analyte stability on DBSs within a 3-month period was monitored. Aliquots of 85 µL of blood from autopsy cases were pipetted onto DBS cards, which were dried and stored at room temperature. DBSs were analyzed in triplicate immediately, within the following 3 weeks, and after 3 months. For each analysis, a whole blood stain was extracted in phosphate buffer and purified using Solid Phase Extraction (SPE) cartridges in order to avoid matrix effects and injected in the LC-MS/MS system. Thirty-nine molecules were screened. Limits of detection (LODs) ranged between 0.1 and 3.2 ng/mL (g) and 0.1 and 5.2 ng/mL (g) for antidepressants and antipsychotics, respectively. Limits of quantification (LOQs) varied from 5 to 10.0 ng/mL for both. Sixteen cases among the 60 analyzed resulted positive for 17 different analytes; for 14 of these the method was fully validated. A general good agreement between the concentrations on DBSs and those measured in conventional blood samples (collected concurrently and stored at -20 °C) was observed. The degradation/enhancement percentage for most of the substances was lower than 20% within the 3-month period. Our results, obtained from real post-mortem cases, suggest that DBSs can be used for routine sample storage.

Ethyl Glucuronide Elimination Kinetics in Fingernails and Comparison to Levels in Hair.

AIMS: Measurement of ethyl glucuronide (EtG) in nail, as a biomarker for alcohol intake, has recently been suggested as alternative to measurement in hair. The aim of this study was to compare levels of EtG in nail and hair, and to investigate the elimination kinetics of EtG in fingernails during an alcohol abstinent period. METHODS: Overall, 40 subjects (median estimated daily intake of ethanol (EDI) 92.5 g/day) were recruited from an alcohol rehabilitation clinic. Nail and hair samples were collected at inclusion and nail clippings were collected every 7-10th day for up to 12 weeks. RESULTS: All patients showed higher nail EtG/EDI ratios compared to hair EtG/EDI ratios (P < 0.001). The median value of the ratios between EtG in nail and EtG in hair was 5.0 (range: 1.07-56.1). There was a significant correlation between nail EtG/EDI and hair EtG/EDI (Spearman's ρ = 0.638, P < 0.001). EtG disappeared from nails after ~2 months of abstinence and the median calculated EtG half-life in nail clippings was 13.3 days (range: 5.5-29.0). There was a significant correlation between the time elapsed to last positive sample for nail EtG and nail EtG levels at time of inclusion (Spearman's ρ = 0.449, P = 0.004). CONCLUSION: The present data indicate that EtG cut-off levels in nails should be higher compared to the established 30 pg/mg EtG cut-off in hair representing heavy drinking. EtG may disappear faster from nail than expected from nail growth physiology. SHORT SUMMARY: Nails are an alternative matrix to hair when measuring ethyl glucuronide (EtG). The present study indicate that EtG cut-off levels in nails should be higher compared to the established 30 pg/mg EtG cut-off in hair representing heavy drinking, and EtG may disappear faster from nail than expected.

Levels of Hair Ethyl Glucuronide in Patients with Decreased Kidney Function: Possibility of Misclassification of Social Drinkers.

BACKGROUND: The use of hair ethyl glucuronide (EtG) levels as a biomarker for chronic high intake of ethanol (EtOH) is increasing, and misclassification of alcohol consumption may have large implications for the patient. The aim of this study was to compare levels of hair EtG in patients with reduced kidney function to levels seen in a comparable control group and to investigate whether the hair EtG levels among kidney failure patients who are social drinkers may lead to a false-positive diagnosis of heavy drinking. METHODS: A total of 41 patients with reduced kidney function and 42 healthy volunteers were included in the study. Both patients and the healthy volunteers reported moderate alcohol intake. The levels of EtG in hair (corrected for estimated daily intake of EtOH [EDI]) were compared between the 2 groups. RESULTS: There was no significant difference between the groups regarding EDI. Despite this, there were significant higher levels of hair EtG (corrected for EDI) in the patient group compared to the control group (p < 0.001). Eight subjects (20%) in the patient group showed EtG levels in hair above 30 pg/mg, in contrast to no subjects among healthy volunteers (p = 0.002). In the patient group, there was significant correlation between levels of EtG in hair and both estimated glomerulus filtration rate and serum creatinine levels. CONCLUSIONS: This study documents an increased risk of obtaining a false-positive diagnosis of heavy drinking among renal disease patients who are social drinkers. Interpretation of EtG levels in hair among patients with reduced kidney function must be performed with caution.

Development of a new immunoassay for the detection of ethyl glucuronide (EtG) in meconium: validation with authentic specimens analyzed using LC-MS/MS. Preliminary results.

BACKGROUND: Ethyl glucuronide (EtG) measurement in neonatal meconium has emerged as a reliable marker to objectively assess prenatal exposure to maternal ethanol complementary to fatty acid ethyl ester (FAEEs) measurement. The detection of EtG in meconium is currently a lengthy, difficult and expensive process using liquid chromatography tandem mass spectrometry (LC-MS/MS) as the analytical procedure. An enzyme-linked immunosorbent assay (ELISA) for the identification of EtG in meconium was developed, validated and applied to authentic meconium specimens from newborns collected in Europe. METHODS: The ELISA procedure was calibrated using 0.45, 0.9, 1.35 and 1.8 nmol/g (100, 200 300 and 400 ng/g) standards. Meconium (0.25 g) was mixed thoroughly, with extraction buffer (pH 7.3; 0.5 mL). The tube was capped, sonicated, centrifuged and the supernatant was decanted. An aliquot of the extract (50 µL) was placed in the well of the microplate followed by enzyme conjugate (150 µL). The plate was incubated for 1 h, washed with deionized water, dried and substrate (200 µL) was added. After 30 min incubation, stop solution was added and the plate was read at 450 nm and 650 nm. Samples were also analyzed for EtG and FAEEs by validated LC-MS/MS assays. RESULTS: Using an EtG cut-off of 0.9 nmol/g for both ELISA screening test and confirmatory LC-MS/MS, immunoassay sensitivity was 100%; specificity 78%; positive-predictive value (PPV) 29% and negative-predictive value (NPV) 100%. CONCLUSIONS: The assay is proposed as a preliminary screening test for the meconium of newborns suspected of being born to mothers drinking alcohol during pregnancy.

Validation of a multi-analyte LC-MS/MS method for screening and quantification of 87 psychoactive drugs and their metabolites in hair.

A multi-analyte method for the detection and quantification of 87 psychoactive drugs (antidepressants, antipsychotics, benzodiazepines, and z-drugs) in human hair has been developed and fully validated using the liquid chromatography-tandem mass spectrometry system. Due to the remarkable increase in requests of hair sample tests (such as for driver's license renewals, child custody, DFA cases, and postmortem toxicology), we focused on the development of a rapid sample preparation. About 20 mg of hair samples, previously washed and cut into snippets, was ultrasonicated with 700 µl of methanol. Samples were then directly analyzed using a 4000 QTRAP (AB SCIEX, Foster City, CA, USA) with an electrospray ionization (ESI) Turbo V(TM) Ion Source. The validation criteria parameters were satisfactory and in accordance with the international guidelines. All the compounds tested were successfully detected. One important aspect is the LODs in the low picogram per milligram concentration which may suggest a potential use of this method in cases of detection of single drug exposure. However, the LC-MS/MS method has been successfully applied for the analysis of postmortem cases (n = 9).

The effects of venovenous bypass use in liver transplantation with piggyback technique: a propensity score-weighted analysis.

Venovenous bypass (VVB) use during liver transplantation (LT) is notably variable among the centres and it is actually restricted to surgically complex cases, severely unstable recipients or grafts from high-risk donors. Historically, VVB was associated with the classical LT with caval cross clamping, while not much is known about the safety of this technique applied to piggyback LT. This retrospective observational study evaluated the effects of VVB applied to piggyback LT on mortality, hospital outcomes, postoperative graft and other organ dysfunction. We retrospectively collected data about recipient status, surgical complexity and graft quality of all the piggyback LTs performed at the Transplant Unit of IRCCS Azienda Ospedaliero-Universitaria di Bologna, Italy, from January 2012 to December 2022. A propensity score (PS) was built taking into account the variables possibly associated with either VVB choice and the investigated outcomes with the average treatment overlap method. PS-weighted general linear models (GLMs) were developed to investigate the adjusted effect of VVB use on the selected outcomes. The final analysis included 874 LT cases, of whom 74 (8.5%) underwent VVB. The effective sample sizes after PS-weighting were 280.2 and 64.3 patients in the no-VVB and VVB groups, respectively. PS-weighted GLMs did not show any differences regarding hospital and graft-related outcomes. However, significantly higher odds ratios for serum creatinine > 2 mg/dL and AKIN stage 2 or 3 during the first 24 h after ICU admission together with a higher renal replacement therapy need during ICU stay were reported for VVB exposure in the weighted analyses. This study suggests similar mortality and length of stay but a higher risk for postoperative acute kidney injury in patients undergoing piggyback LT with VVB.

Higher levels of hair ethyl glucuronide in patients with decreased kidney function.

BACKGROUND: Hair levels of ethyl glucuronide (EtG) are often used to differentiate social drinking from heavy drinking. Patients with decreased kidney function have delayed excretion of EtG, and increased incorporation into hair could be suspected. The aim of this study was to compare hair EtG levels in patients with decreased kidney function to those seen in healthy volunteers. METHODS: Twelve patients with renal disease were included. The levels of EtG in hair were adjusted to estimated daily intake of ethanol (EDI) and compared to 21 previously published healthy individuals. RESULTS: The levels of hair EtG in the 12 patients ranged between < limit of detection and 134 pg/mg, and the EDI ranged between 0.1 and 12 g. The levels of EtG in hair were significantly higher in the patients compared to healthy volunteers (p = 0.009). CONCLUSIONS: These preliminary results indicate that hair levels of EtG in a population of patients with decreased kidney function should be interpreted with caution.

Testing ethylglucuronide in maternal hair and nails for the assessment of fetal exposure to alcohol: comparison with meconium testing.

BACKGROUND: The deleterious effects exerted by prenatal ethanol exposure include physical, mental, behavioral, and/or learning disabilities that are included in the term fetal alcohol spectrum disorder. The measurement of ethylglucuronide (EtG) in alternative biological matrices, including neonatal and maternal hair, neonatal meconium, and maternal nails, is receiving increasing interest for the accurate evaluation of the in utero exposure to alcohol. OBJECTIVE: To evaluate the correlation between EtG in maternal hair and nails with EtG in neonatal meconium to further explore the suitability of these biomarkers in disclosing prenatal exposure to ethanol. METHODS: A total of 151 maternal hair strands (0-6 cm), nail clips (2-6 mm), and corresponding neonatal meconium and nails samples were obtained from neonatal wards of 4 Mediterranean public hospitals: Rome, Florence, and Belluno in Italy and Barcelona in Spain. Hair, nails, and meconium were analyzed for the presence of EtG by validated liquid chromatography mass spectrometry assay. Meconium was also analyzed for the presence of fatty acid ethyl esters (FAEEs) as a complementary biomarker of potential in utero exposure to alcohol. RESULTS: Eighteen newborns resulted in utero exposed to maternal alcohol consumption by FAEE testing in meconium with EtG values between 0.5 and 1.5 nmol/g. Unfortunately, none of these cases were confirmed by the presence of EtG in maternal hair and nails samples, which resulted all negative to this biomarker. DISCUSSION AND CONCLUSIONS: The results confirm that FAEEs and EtG in meconium are the best biomarkers to assess in utero exposure to maternal alcohol. EtG in hair and nails are not good biomarkers to disclose alcohol consumption lower than on daily basis and lower than 1-2 alcoholic units per day.

Survey on health students' knowledge and perception on body donation for scientific research, education, and training after specific Italian law no. 10/2020.

BACKGROUND: Practicing on the human body was considered extremely relevant for health professionals' education, but a drastic reduction was observed due to an increase in alternative virtual and multimedia means, and, in Italy, also due to a lack of regulation. Italian Law 10/2020 regulates body donation for research and training through an advanced directive for post-mortem body donation. METHODS: A cross-sectional study was carried out to investigate the law knowledge and body donation perception of health students of any degree courses enrolled at the University of Pavia, Italy, in 2021, through ad hoc web questionnaire. RESULTS: 485 students participated to this survey; median age was of 21 years (25th-75th percentiles, 20-23), 73.2% were females, and 62.5% were medical students. Among them 14.9% knew the Italian law 10/2020. Age was the only variable associated with students' knowledge of the law. Further, 8.3% reported the current availability of cadaveric practice, 85.6% of health students acknowledged usefulness of cadaveric practice, with a significant difference between medical and non-medical students (71.4% vs 28.6%, p < 0.001). Overall, 59.7% would donate their body, rising to 62.7% with reference to specific law regulation, with 30.5% and 28.7% undecided, respectively. 51.3% of participants answered not practicing religious faith, 82.9% with Catholic families, without significance on the knowledge of the law. CONCLUSIONS: Poor knowledge of the law compared with great interest and acknowledged cadaveric practice usefulness highlighted the need for better information, especially among health students, where critical discussion could be more valuable. Then, there arises the urgent need to fill the gaps within university studies and syllabuses, to relaunch the central role that cadaveric practice and research had in the education of health professionals. Consequences on basic and specific health students' skills, on health education quality in general, and further on health professionals' expertise must be carefully considered.

Assessment of prenatal exposure to ethanol by meconium analysis: results of an Italian multicenter study.

BACKGROUND: This study estimated in 7 Italian cities the prevalence of prenatal exposure to ethanol by determining fatty acid ethyl esters (FAEEs; palmitic, palmitoleic, stearic, oleic, linoleic, linolenic, and arachidonic esters) and ethyl glucuronide (EtG) in neonatal meconium samples. METHODS: A total of 607 meconium samples were obtained from neonatal wards of 7 public hospitals: Verona and San Daniele del Friuli in the northeast of the country, Reggio Emilia in the middle east, Florence and Rome in the center, and Naples and Crotone in the southwest of the peninsula. Meconium biomarkers were assessed by a validated methodology using liquid chromatography-tandem mass spectrometry and the results categorized using the accepted cutoff of 2 nmol/g total amount of 7 FAEEs and 2 nmol/g EtG, to differentiate between heavy maternal ethanol use during pregnancy and occasional or no use at all. RESULTS: On the basis of the above-reported cutoffs, the overall prevalence of newborns prenatally exposed to maternal ethanol was 7.9%: 0% in Verona, 4.0% in San Daniele del Friuli, 4.9% in Naples, 5.0% in Florence, 6.2% in Crotone, up to 10.6% in Reggio Emilia, and 29.4% in Rome. Low maternal education level and younger maternal age were associated with biomarker scores over the cutoff. There was also a significant correlation between the highest percentage of prenatal exposure in the capital and certain maternal sociodemographic characteristics. CONCLUSIONS: These results indicate considerable variability in the prevalence of fetal exposure to ethanol in different Italian cities, as determined by the objective measurement of biomarkers in meconium. These data, together with previous ones obtained in Barcelona, Spain, indicate that gestational ethanol exposure is widespread, at least in parts of Europe.

Determination of ethyl glucuronide in nails by liquid chromatography tandem mass spectrometry as a potential new biomarker for chronic alcohol abuse and binge drinking behavior.

A liquid chromatography tandem mass spectrometry method for ethyl glucuronide (EtG) detection and quantification in nails was developed and fully validated. Nails were extracted in 700 µL double-distilled water. EtG-d(5) was used as an internal standard. Reversed-phase separation was obtained with an isocratic mobile phase composed of 0.1% formic acid and acetonitrile (99:1) for 10 min. Quantification was performed by multiple reaction monitoring of two transitions per compound (EtG and internal standard). The assay was linear from 10 to 500 pg/mg. Validation parameters were studied at three different quality control levels (10, 50, and 300 pg/mg). Intraday, interday, and total imprecision had a coefficient of variation of less than 9.5%. Ion suppression and ion enhancement were negligible (less than 20%). No carryover was detected. The method was applied to several real cases, among teetotalers, social drinkers, and heavy drinkers. A questionnaire, together with the informed consent form, was given to all the participants in order to evaluate alcohol intake in the one month before sample collection. Nail EtG levels in a social drinker were much higher than the concentrations of EtG in hair provided by the same subject, thus suggesting potential high sensitivity in evaluating both chronic excessive alcohol consumption and binge drinking habits.

Cocaine use during pregnancy assessed by hair analysis in a Canary Islands cohort.

BACKGROUND: Drug use during pregnancy is difficult to ascertain, and maternal reports are likely to be inaccurate. The aim of this study was to estimate the prevalence of illicit drug use among pregnant women by using maternal hair analysis. METHODS: A toxicological analysis of hair was used to detect chronic recreational drug use during pregnancy. In 2007, 347 mother-infant dyads were included from the Hospital La Candelaria, Santa Cruz de Tenerife, Canary Islands (Spain). Data on socioeconomic characteristics and on substance misuse during pregnancy were collected using a structured questionnaire. Drugs of abuse: opiates, cocaine, cannabinoids and amphetamines were detected in maternal hair by immunoassay followed by gas chromatography-mass spectrometry for confirmation and quantitation. RESULTS: Hair analysis revealed 2.6% positivity for cocaine and its metabolites. Use of cocaine during pregnancy was associated with unusual behaviour with potentially harmful effects on the baby. CONCLUSIONS: The results of the study demonstrate significant cocaine use by pregnant women in Canary Islands. The data should be used for the purpose of preventive health and policy strategies aimed to detect and possibly to avoid in the future prenatal exposure to drugs of abuse.

Ethyl glucuronide in hair: A 5-year retrospective cohort study in subjects sanctioned for driving under the influence of alcohol and psychoactive substances.

The evaluation of drinking behaviors can help in limiting high-risk situation, such as driving under the influence (DUI). We investigate ethyl glucuronide in hair (hEtG) levels to evaluate alcohol consumption behavior in subjects followed up after having been charged for DUI of psychoactive substances and/or alcohol. We performed a retrospective observational cohort study on 4328 subjects over 18 years old who underwent hEtG analysis in the period 2015-2019 in the Italian Province of Pavia. hEtG level was used as a proxy for the alcohol consumption behavior. Effects of age, sex, and district on alcohol drinking behavior were investigated with an ordinal logit model. A state sequence analysis was used to study people's alcohol consumption behavior over time. hEtG was found ≥7.0 pg/mg in 22.2% of the drivers (of which 7% has an hEtG ≥30.0 pg/mg). Among positive cases, a prevalence of males (96.3%) aged 35-44 (32.6%), coming from main city and hinterland (38.2%), was observed. The propensity to drink was higher for males (odds ratio [OR] ≈ 2.28, p < 0.001) and for subject coming from the district devoted to the cultivation of vineyards. Young age classes have a reduced drinking risk if compared to the drivers over 55 years old (p < 0.001). A general decreasing trend over time in hEtG values was observed. Being male, age ≥ 55 years, and coming from rural areas are potential risk factors related to alcohol drinking habits among drivers. Ethyl glucuronide in hair test in the driving license reissuing protocol contributed to decrease alcohol misuse behaviors.

Determination of Traditional and Designer Benzodiazepines in Urine through LC-MS/MS.

BACKGROUND: The detection of new designer benzodiazepines in biological fluids and tissues, together with the traditional ones, could represent an important analytical update for laboratories performing clinical and forensic toxicological analysis. OBJECTIVE: A liquid chromatography tandem mass spectrometry method (LC-MS/MS) has been developed, fully validated, and applied to a cohort of real urine samples collected from patients under withdrawal treatment and from intoxication cases. METHODS: 100 µL urines were added to a buffer solution containing deuterated internal standards; the samples were then extracted through a liquid/liquid procedure, dried under a nitrogen stream, and reconstituted in mobile phase. The chromatographic separation was performed in reverse phase through a C18 column with gradient elution. Mass spectrometry operated in positive polarization and multiple reaction monitoring mode. RESULTS: 25 molecules were optimized for instrumental analysis: 9 designer benzodiazepines and 16 traditional compounds (parent drugs and main metabolites). Sensitivity, specificity, linearity, accuracy, imprecision, recovery, matrix effects, and carry-over have been evaluated for all molecules. Only cinazepam did not satisfy all acceptance criteria for validation. 10 among the 50 analyzed samples tested positive for at least one of the monitored molecules. In particular, two different samples collected from the same case provided positive results for flubromazepam, a designer benzodiazepine. CONCLUSION: The method was proven to be useful in detecting not only traditional benzodiazepines but also new designer ones. The identification of a New Psychoactive Substance in real samples confirmed that analytical procedures should be updated to include as many substances as possible.

Resveratrol and Its Analogue 4,4'-Dihydroxy-trans-stilbene Inhibit Lewis Lung Carcinoma Growth In Vivo through Apoptosis, Autophagy and Modulation of the Tumour Microenvironment in a Murine Model.

Lung cancer is the most prevalent cancer worldwide. Despite advances in surgery and immune-chemotherapy, the therapeutic outcome remains poor. In recent years, the anticancer properties of natural compounds, along with their low toxic side effects, have attracted the interest of researchers. Resveratrol (RSV) and many of its derivatives received particular attention for their beneficial bioactivity. Here we studied the activity of RSV and of its analogue 4,4'-dihydroxystilbene (DHS) in C57BL/6J mice bearing cancers resulting from Lung Lewis Carcinoma (LLC) cell implantation, considering tumour mass weight, angiogenesis, cell proliferation and death, autophagy, as well as characterization of their immune microenvironment, including infiltrating cancer-associated fibroblasts (CAFs). C57BL/6J mice started treatment with RSV or DHS, solubilised in drinking water, one week before LLC implantation, and continued for 21 days, at the end of which they were sacrificed, and the tumour masses collected. Histology was performed according to standard procedures; angiogenesis, cell proliferation and death, autophagy, infiltrating-immune cells, macrophages and fibroblasts were assessed by immunodetection assays. Both stilbenic compounds were able to contrast the tumour growth by increasing apoptosis and autophagy in LLC tumour masses. Additionally, they contrasted the tumour-permissive microenvironment by limiting the infiltration of tumour-associated immune-cells and, more importantly, by counteracting CAF maturation. Therefore, both stilbenes could be employed to synergise with conventional oncotherapies to limit the contribution of stromal cells in tumour growth.