A Sensitive Liquid Chromatography-Tandem Mass Spectrometry Method for Measuring Fosfomycin Concentrations in Human Prostatic Tissue.

The aim of this study was to develop and validate a fast and sensitive bioanalytical method for the accurate quantification of fosfomycin concentrations in human prostatic tissue. The sample preparation method only required milligrams of tissue sample. Each sample was mixed with two times its weight of water and homogenized. A methanol solution that was three times the volume of the internal standard (fosfomycin-13C3) was added, followed by vortex mixing and centrifugation. After its extraction from the homogenized prostatic tissue, fosfomycin was quantified by means of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) triple quadrupole system operating in negative electrospray ionization and multiple reaction monitoring detection mode. The analytical procedure was successfully validated in terms of specificity, sensitivity, linearity, precision, accuracy, matrix effect, extraction recovery, limit of quantification, and stability, according to EMA guidelines. The validation results, relative to three QC levels, were 9.9% for both the within-day and inter-day accuracy (BIAS%); 9.8% for within-day precision; and 9.9 for between-day precision. A marked matrix effect was observed in the measurements but was corrected by normalization with the internal standard. The average total recovery was high (approximatively 97% at the three control levels). The dynamic range of the method was 0.1-20 µg/g (R2 of 0.999). Negligible carry-over was observed after the injection of highly concentrated samples. F in the sample homogenate extracts was stable at 10 °C and 4 °C for at least 24 h. In the tissue sample freeze-thaw experiments, a significant decrease in F concentrations was observed after only two cycles from -80 °C to room temperature. The novel method was successfully applied to measure fosfomycin in prostatic tissue samples collected from 105 patients undergoing prostatectomy.

Validation and application of a method for the quantification of 137 drugs of abuse and new psychoactive substances in hair.

INTRODUCTION: In the dynamic universe of new psychoactive substances (NPS), the identification of multiple and chemically diverse compounds remains a challenge for forensic laboratories. Since hair analysis represents a gold-standard to assess the prevalence of NPS, which are commonly detected together with classical drugs of abuse (DoA), our study aimed at developing a wide-screen method to detect and quantify 127 NPS and 15 DoA on hair. MATERIALS AND METHODS: A multi-analyte ultra-high performance liquid chromatography mass spectrometry method for the identification and quantification of 127 NPS (phenethylamines, arylcyclohexylamines, synthetic opioids, tryptamines, synthetic cannabinoids, synthetic cathinones, designer benzodiazepines) and 15 DoA in hair samples was developed. A full validation was performed according to the European medicines Agency (EMA) guidelines, by assessing selectivity, linearity, accuracy, precision, limit of quantification (LOQ), limit of detection (LOD), matrix effect and recovery. As a proof of the applicability, the method was applied to 22 authentic hair samples collected for forensic purposes. RESULTS: Successful validation was achieved, by meeting the required technical parameters, for 137 compounds (122 NPS and 15 DoA), with LOQ set at 4 pg/mg for 129 compounds, at 10 pg/mg for 6 and at 40 pg/mg for 2. The method was not considered validated for 5 NPS, as LLOQ resulted too high for a forensic analysis (80 pg/mg). Among authentic forensic samples, 17 tested positive for DoA, and 10 to NPS, most samples showing positivity for both. Detected NPS were ketamine and norketamine, 5-MMPA, ritalinic acid, methoxyacetyl fentanyl, methylone and RCS-4. CONCLUSION: The present methodology represents an easy, low cost, wide-panel method for the quantification of 122 NPS and 15 DoA, for a total of 137 analytes, in hair samples. The method can be profitably applied by forensic laboratories. Similar multi-analyte methods on the hair matrix might be useful in the future to study the prevalence of NPS and the co-occurrence of NPS-DoA abuse.

Fast and Sensitive Analysis of Fosfomycin in Human Plasma Microsamples Using Liquid Chromatography-Tandem Mass Spectrometry for Therapeutic Drug Monitoring.

BACKGROUND: Fosfomycin is an antibiotic recently repurposed as a potential combination treatment for difficult-to-treat Gram-negative bacterial infections. The pharmacokinetic features of fosfomycin have demonstrated that different pathophysiologic alterations may affect its exposure. Therapeutic drug monitoring may improve real-time management of fosfomycin therapy in different clinical scenarios. OBJECTIVES: To develop and validate a fast and sensitive liquid chromatography - tandem mass spectrometry method for measuring fosfomycin in human plasma microsamples (3 µL). METHODS: Analysis was preceded by a user-friendly pre-analytical single-step process performed via a rapid chromatographic run of 2.5 minutes, followed by negative electrospray ionization and detection on a high-sensitivity triple quadrupole tandem mass spectrometer operated in the multiple reaction monitoring mode. European Medicines Agency guidelines were used to validate the specificity, sensitivity, linearity, precision, accuracy, matrix effects, extraction recovery, limits of quantification, and stability of the analytical method. RESULTS: The new assay produced accurate (BIAS%: 0.9-9.1) and precise (coefficient of variation [CV]%: 8.1-9.5) measurements of fosfomycin over a concentration range of 1-1000 mg/L. Overall, analyte recovery was consistent (mean values: 91.2%-97.2%) at all tested concentration levels. The analyte was also stable in human plasma and the final extract under various storage conditions. The clinical applicability of the assay was confirmed through quantitation of plasma samples obtained from patients. CONCLUSIONS: A sensitive liquid chromatography - tandem mass spectrometry method for measuring fosfomycin in plasma was developed and validated according to the European Medicines Agency criteria. Quantitation of fosfomycin in clinical plasma samples confirmed that the assay is suitable for therapeutic drug monitoring in clinical scenarios.

Fast and Sensitive Method for Simultaneous Quantification of Meropenem and Vaborbactam in Human Plasma Microsamples by Liquid Chromatography-Tandem Mass Spectrometry for Therapeutic Drug Monitoring.

Meropenem (MRP)-Vaborbactam (VBR) is a novel beta-lactam/beta-lactamase inhibitor used for the management of difficult-to-treat Gram-negative infections. Among critically ill patients, MRP-VBR shows remarkable inter-individual variability in pharmacokinetic behavior, thus justifying the implementation of therapeutic drug monitoring (TDM) for improving real-time management in different challenging scenarios. In this study, we developed and validated a fast and sensitive Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) method for the simultaneous quantification of MRP and VBR in human plasma microsamples of 3 microliters. The analysis required only a single-step sample preparation and was performed by means of a fast chromatographic run of 4 min, followed by positive electrospray ionization and detection on a high-sensitivity triple quadrupole tandem mass spectrometer operated in multiple reaction monitoring modes. The straightforward analytical procedure was successfully validated, based on the EMA guidelines, in terms of specificity, sensitivity, linearity, precision, accuracy, matrix effect, extraction recovery, the limit of quantification, and stability. The novel method was successfully applied for simultaneously measuring MRP and VBR concentrations in more than 42 plasma samples collected from critically ill patients affected by carbapenem-resistant Gram-negative bacteria infections.

Development and validation of a fast UPLC-MS/MS screening method for the detection of 68 psychoactive drugs and metabolites in whole blood and application to post-mortem cases.

We report a rapid and sensitive LC-MS/MS method that allows the simultaneous detection of 68 commonly prescribed antidepressants, benzodiazepines, neuroleptics, and metabolites in whole blood with a small sample volume after a rapid protein precipitation. The method was also tested on post-mortem blood from 85 forensic autopsies. Three sets of commercial serum calibrators containing a mix of prescription drugs of increasing concentration were spiked with red blood cells (RBC) to obtain 6 calibrators (3 "serum calibrators" and 3 "blood calibrators"). Curves obtained from serum calibrators and from blood calibrators were compared using a Spearman correlation test and by analyzing slopes and intercepts, to assess if the points from six calibrators could be plotted together in a single calibration model. The validation plan included interference studies, calibration model, carry-over, bias, within-run and between-run precision, limit of detection (LOD), limit of quantification (LOQ), matrix effect and dilution integrity. Four deuterated Internal Standards (Nordiazepam-D5, Citalopram-D6, Ketamine-D4 and Amphetamine-D5) and two different dilutions were assessed. Analyses were performed using an Acquity UPLC® System coupled with triple quadrupole detector Xevo TQD®. The degree of agreement with a previously validated method was calculated on whole blood samples of 85 post-mortem cases, by performing a Spearman correlation test with a Bland-Altman plot. Percentage error between the two methods was evaluated. Slopes and intercepts of curves obtained from serum calibrators and from blood calibrators showed a good correlation, and the calibration model was built plotting all points together. No interferences were found. The calibration curve appeared to provide a better fit of the data using an unweighted linear model. Negligible carry-over was observed, and very good linearity, precision, bias, matrix effect and dilution integrity were achieved. The LOD and the LOQ were at the lower limits of the therapeutic range for the tested drugs. In a series of 85 forensic cases, 11 antidepressants, 11 benzodiazepines and 8 neuroleptics were detected. For all analytes, a very good agreement between the new method and the validated method was demonstrated. The innovation of our method consists in the use of commercial calibrators, readily available to most forensic toxicology laboratories, for the validation of a fast, inexpensive, wide-panel LC-MS/MS method that can be used as a reliable and accurate screening for psychotropic drug in postmortem samples. As observed in the implementation on real cases, this method could be profitably applied in forensic cases.

Fast and Simple Liquid Chromatography-Isotope Dilution Tandem Mass Spectrometry Method for Therapeutic Drug Monitoring of Dalbavancin in Long-Term Treatment of Subacute and/or Chronic Infections.

Dalbavancin (DBV) is a long-acting antistaphylococcal lypoglycopeptide that is being increasingly used for long-term treatment of a wide range of subacute and/or chronic infections, mainly osteo-articular infections (OAI). Population pharmacokinetic studies showed that two 1500 mg doses 1 week apart can ensure effective treatment for several weeks. In this scenario, therapeutic drug monitoring (TDM) can be a helpful tool for providing clinicians with real-time feedback on the duration of optimal treatment by measuring drug concentrations over time in each single patient. The aim of this study was to develop and validate a fast and simple analytical method based on the Liquid Chromatography-Isotope Dilution Tandem Mass Spectrometry (ITD LC-MS/MS) technique for measuring DBV concentrations in human plasma microsamples. It will allow an innovative, very convenient and minimally invasive way of sampling. Analysis was performed by simple single-step sample preparation and very short instrumental run time (4 min). Analytical performance met all criteria in terms of specificity, sensitivity, linearity, precision, accuracy, matrix effect, extraction recovery, limit of quantification, dilution integrity and stability under different conditions set by the European Medicines Agency (EMA) for drug quantification by means of bioanalytical methods. The method was successfully applied for measuring DBV concentrations (range = 2.0-77.0 mg/L) in a cohort of patients receiving long-term DBV treatment of subacute and/or chronic infections.

Fast and Sensitive Analysis of Cefiderocol in Human Plasma Microsamples by Liquid Chromatography-Isotope Dilution Tandem Mass Spectrometry for Therapeutic Drug Monitoring.

Cefiderocol (C) is a parenteral siderophore cephalosporin with relevant inter-individual pharmacokinetic variability among critically ill patients, which may potentially affect effective drug exposure. Therapeutic drug monitoring (TDM) may concur in improving the real-time management of C therapy in clinics. In this study, we developed and validated a fast and sensitive Liquid Chromatography-Isotope Dilution Tandem Mass Spectrometry (LC-ITD-MS/MS) method for measuring C in human plasma microsamples, as small as 3 microliters. Analysis was preceded by a user-friendly pre-analytical single-step and was performed by means of a very fast chromatographic run of 4 min, followed by positive electrospray ionization and detection on a high sensitivity triple quadrupole tandem mass spectrometer operated in multiple reaction monitoring mode. The straightforward analytical procedure was successfully validated, based on the European Medicines Agency (EMA) guidelines, in terms of specificity, sensitivity, linearity, precision, accuracy, matrix effect, extraction recovery, limit of quantification, and stability. The novel method was successfully applied to TDM of C in more than 50 cases of critically carbapenem-resistant Gram-negative bacterial infections and enabled us to optimize antibiotic therapy.

Valproic acid determination by liquid chromatography coupled to mass spectrometry (LC-MS/MS) in whole blood for forensic purposes.

Valproic acid (VPA) is a well-known drug prescribed as anti-epileptic. It has a narrow therapeutic range and shows great individual differences in pharmacodynamics and pharmacokinetics. Consequently, the therapeutical drug monitoring (TDM) in patient's plasma is of crucial importance. Liquid chromatography coupled to mass spectrometry (LC-MS/MS) has gained importance in TDM applications for its features of sensitivity, selectivity and rapidity. However, in case of VPA, the LC-MS/MS selectivity could be hampered by the lack of a sufficient number of multiple reaction monitoring (MRM) transitions describing the molecule. In fact, the product ion scan of deprotonated molecules of VPA does not produce any ion and thus most LC-MS/MS methods are based on the detection of the unique MRM transition m/z 143➔143. In this way, the advantages of selectivity in LC-MS cannot be effectively exploited. In the present method, stable analyte adducts were exploited for the determination of VPA in blood. An Acquity HSS C18 column and mobile phases consisting of 5-mM ammonium formate and acetonitrile both added 0.1% formic acid were used. Source worked in negative acquisition mode and parameters were optimized to increase the adduct (m/z 189) and dimer (m/z 287) stability, and their fragmentation were used to increase the selectivity of MRM detection. The method has been validated according to the toxicological forensic guidelines and successfully applied to 10 real blood samples. Finally, the present method showed suitable for the rapid LC-MS/MS detection of VPA in whole blood, demonstrating the possibility to increase specificity by exploiting stable in-source adducts. This should be considered of utmost importance in the case of forensic applications.

Gender differences in driving under the influence of psychoactive drugs: Evidence mapping of real case studies and meta-analysis.

BACKGROUND: Very few studies have examined predictors of Driving Under the Influence (DUI) as a function of gender. This oversight is relevant, because analyzing gender differences prevents generalization of results observed in men, who still currently account for the majority of drivers worldwide, to women. The aim of this study is to analyze the prevalence of DUI of drugs in men and women reported in real case studies published in the last two decades, and to assess gender differences in risky DUI behaviour. METHODS: PubMed, Scopus, Web of Science were searched for eligible studies in May 2021; a follow-up literature search was conducted in August 2021. Real-case studies of drivers convicted for DUI of psychoactive drugs with positive toxicological confirmatory analysis were included. The extracted outcome was the prevalence of positive findings of men and women for cocaine, cannabinoids, amphetamine-like drugs, opioids, and psychoactive prescription drugs. A meta-analysis of random effects estimates was performed to investigate the change in the size of the overall effect (by Cohen d standardized mean difference test). A Mann Whitney U test was performed to test for differences between genders. RESULTS: Of the 2877 studies screened, 439 were retrieved in full-text and 26 were included. The meta-analysis showed a significant higher prevalence among men for cocaine (1.8% vs 0.9%; p < 0.001), cannabinoids (3.5% vs 1.6%; p = <0.01) and amphetamine-like drugs (1.2% vs 0.6%; p < 0.01). Surprisingly, no differences were observed in the use of opioids (2.3% vs 2.2%; p = 0.45) and benzodiazepines/Z-drugs (2.9% vs 3.7%; p = 0.52). CONCLUSION: Contrary to the extraordinary number of real-case studies reported in literature, only a few papers differentiate the prevalence of DUI between men and women. This can lead to an underestimation of the influence of gender in DUI phenomenon or complicate the evaluation of the results for some classes of substances, as observed for medications and opioids. The primary goal in the future will be to collect the data concerning DUI drivers following shared and homogeneous methodologies, in order to allow the analysis of data disaggregated by gender, which can be used for monitoring evolving trends and developing gender-specific targeted prevention and enforcement efforts.

The use of fly artifacts in a crime scene: Is there any application for forensic toxicology?

Fly artifacts (FA) are bloodstains resulting from insect activity at a crime scene, usually by feeding on human blood. Whether these artifactual stains might be useful for forensic toxicological investigations in cases of absence of conventional and unconventional matrices, for example, in cases concealment of the body or of extensive putrefaction, has not yet been investigated. The purpose of this study is to understand if FA trace evidence permits toxicological analysis when traditional matrices are not available. To this aim, FA experimentally produced by Calliphora vomitoria feeding on human blood of a cocaine and heroin user were collected from absorptive and non-absorptive material. FA material was analyzed by a new simple and fast LC-MS/MS method. Results were evaluated in terms of presence of the drug and relative amount of the detected molecules. From a qualitative point of view, the analysis of FA revealed all the substances originally detected in post-mortem blood in both cases. The ratios of cocaine/benzoylecgonine, codeine/morphine, and 6-monoacetylmorphine/morphine recovered in FA from cotton-textile materials and from non-absorptive surfaces were consistent with data resulted from original post-mortem blood. The preliminary study herein reported demonstrated that FA are extremely informative in case of cocaine and heroin users and merit further research in order to be applied in real caseworks.

Development and validation of a rapid LC-MS/MS method for the detection of 182 novel psychoactive substances in whole blood.

INTRODUCTION: The analysis of novel psychoactive substances (NPS) represents a challenge in forensic toxicology, due to the high number of compounds characterized by different structures and physicochemical properties both among different subclasses and within a single subclass of NPS. The aim of the present work is the development and validation of a targeted liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the detection of NPS in whole blood. MATERIALS AND METHODS: A protein-precipitation based LC-MS/MS method for the detection of more than 180 NPS was developed and validated by assessing the following parameters: selectivity, linearity, accuracy, precision, limit of detection (LOD) and of quantification (LOQ) recovery, and matrix effect. Then, the method was applied to real forensic samples. RESULTS: The method allowed the identification of 132 synthetic cannabinoids, 22 synthetic opioids, and 28 substances among synthetic cathinones, stimulants, and other drugs. Validation was successfully achieved for most of the compounds. Linearity was in the range of 0.25-10 ng/ml for synthetic cannabinoids and 0.25-25 ng/ml for other drugs. Accuracy and precision were acceptable according to international guidelines. Three cases tested positive for fentanyl and ketamine, in the setting of emergency room administration. CONCLUSIONS: The present methodology represents a fast, not expensive, wide-panel method for the analysis of more than 180 NPS by LC-MS/MS, which can be profitably applied both in a clinical context and in postmortem toxicology.

"Light cannabis" consumption in a sample of young adults: Preliminary pharmacokinetic data and psychomotor impairment evaluation.

INTRODUCTION: In 2019, the Italian Supreme Court established that hemp cannot be commercialized for human use, when the "psychotropic effect" of the product or its "offensiveness" can be demonstrated. The aim of the present study is to assess Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD) blood concentrations after smoking cannabis with a low percentage of Δ9-THC, also referred as "light cannabis", and its effects on young adults' vigilance, cognitive and motor skills. MATERIALS AND METHODS: Eighteen young adults consumed three light cannabis cigarettes containing 400 mg of inflorescences each, with a percentage of 0.41% of Δ9-THC and of 12.41% of CBD. Blood samples were collected before the experiment (t0), after each light cannabis cigarette (t1→t3), 60 (t4) and 120 (t5) minutes after the beginning of the experiment. Five performance tasks and a subjective scale were employed for measuring cognitive and psychomotor performances the day before the experiment (TT0) and after the third cigarette (TT1). RESULTS: Mean (SD) concentrations (ng/ml) were 1.0 (0.8) in t1, 1.2 (0.9) in t2, 1.0 (0.8) in t3, 0.6 (0.4) in t4 and 0.3 (0.3) in t5 for Δ9-THC; 10.5 (10.3) in t1, 10.3 (13.2) in t2, 15.1 (14.8) in t3, 9.9 (9.2) in t4 and 5.7 (5.7) in t5 for CBD. No significant differences were observed between TT0 and TT1 for all performed psychomotor performance task. None of the subjects declared to feel "high" after the experiment. DISCUSSION: All study participants reported that a higher number of cigarettes, corresponding in this study to 1200 mg of herbal product, could hardly be consumed by smoking in a recreational setting. Δ9-THC and CBD concentrations showed a high inter-subject variability, and the average concentrations were lower than those previously reported. Toxicological results showed a decrease of Δ9-THC and CBD after the third light cannabis cigarette, and a Δ9-THC /CBD ratio always<1 was observed. The lack of impairment observed in our participants can be interpreted as a consequence of the very low concentrations detectable in the blood.

Deaths related to nitrogen inhalation: Analytical challenges.

Dinitrogen (N2) has been increasingly connected to suicidal deaths. The analysis of N2 in post-mortem cases still represents a major challenge in forensic toxicology and circumstantial data has so far played a major role for the determination of the cause of death. In this paper, after presenting a review of cases of N2 intoxication described in forensic literature, we report the application of two approaches in order to quantify an excess of N2 in post-mortem whole blood collected from a case of suicide by nitrogen inhalation. N2 analyses were performed by GC-MS on the suicidal case and on controls taken from 10 autopsy cases with similar PMI (5 traumatic deaths and 5 deaths by asphyxia). The percentage of N2 was estimated by building a five-point N2 peak area calibration curve (0, 15.6 %, 62.4 % 78.1 %, 100 %) and through an external QC, assessing linearity, accuracy and precision, LLOQ, specificity and stability of N2 in the sample vial. Percentage of N2 of the case was significantly higher than the post-mortem controls (p<0.05). The N2/O2 ratio of the case and controls was also calculated as an additional indicator, and was significantly higher in the case (p<0.05). The strengths and the limitation of both methods are reported in the paper. Toxicological confirmation for N2 are rarely performed when the cause of death is evident, probably due to the lack of validated methods and the complexity of the interpretation of N2 concentration in biological fluids. The presented methods can be rapidly and profitably applied with instrumentation normally available in forensic laboratories.

Optimization of cloned enzyme donor immunoassay cut-offs for drugs of abuse in post-mortem whole blood.

INTRODUCTION: Immunoassay (IA) tests are not widely applied in post-mortem samples, since they are based on technologies requiring relatively non-viscous specimens, and compounds originating from the degradation of proteins and lipids during the post-mortem interval can alter the efficiency of the test. However, since the extraction techniques for IA tests are normally rapid and low-cost, IA could be used as near-body drug-screening for the classes of drugs most commonly found in Italy and Europe. In this study, semi-quantitative results on post-mortem whole blood samples obtained through CEDIA analysis (cannabinoids, cocaine, amphetamine compounds, opiates and methadone), were compared with results of confirmatory analysis obtained using GC-MS. Screening cut-offs for all drugs were retrospectively optimized. METHODS: Post-mortem whole blood samples from autopsy cases of suspected fatal intoxication were collected over 3 years. Samples were initially analyzed through CEDIA (CEDIA, ILab 650, Werfen). Confirmatory analyses were then performed by GC-MS (QP 2010 Plus, Shimadzu). Screening cut-offs were retrospectively optimized using Receiver Operating Characteristic (ROC) analysis. RESULTS: CEDIA results were available for 125 samples. Two-hundred-eighty-nine (289) positive screening results were found. Among these, 162 positive confirmation results were obtained. Optimized screening cut-offs were as follows: 6.5ng/ml for THC; 4.2ng/ml for THC-COOH; 12.0ng/ml for cocaine; 6.6ng/ml for benzoylecgonine; 6.4ng/ml for opiates; 2.0ng/ml for methadone. Analysis of ROC-curves showed a satisfying degree of separation in all tests except for amphetamine compounds, with areas under the curve (AUC) between 0.915 (THC) and 0.999 (for benzoylecgonine and methadone). DISCUSSION: The results of the study showed that CEDIA screening at the optimized cut-offs exhibits a very high sensitivity and good specificity and positive predictive value (PPV) for cannabinoids, cocaine and metabolites, opiates and methadone. A high number of false positives (n=19) for amphetamine compounds was observed at the optimized cut-off, resulting in a very low PPV, which is also influenced by the very low number of TP (n=4). CONCLUSION: The results of the study show that the CEDIA is a valuable screening test on post-mortem whole blood for cannabinoids, cocaine and metabolites, opiates and methadone, but it is not recommended for amphetamine compounds, due to the high number of false positives. The strengths of the study are the large sample size, the inclusion of post-mortem cases only and the high level of sensitivity and specificity obtained at the optimized cut-offs.

Alcohol and illicit drugs in drivers involved in road traffic crashes in Italy. An 8-year retrospective study.

This study aims to investigate the prevalence of alcohol and drugs of abuse in Italian drivers involved in road traffic crashes between 2011 and 2018. Toxicological analyses were performed on the whole blood of 7593 injured drivers. Alcohol and illicit drugs, namely tetrahydrocannabinol (THC; cut-off 2ng/ml), cocaine (cut-off 10ng/ml), illicit opiates (cut-off 10ng/ml) and amphetamines (amphetamine, methamphetamine, MDMA, MDA; cut-off 20ng/ml) were investigated. The age and gender of the driver, the time of the crash (weekend/weekday and day/night), the road crash year and Blood Alcohol Concentration (BAC) were also considered. The 16.2% of samples tested positive for alcohol, 2.5% for cocaine, followed by opiates (2.0%), cannabinoids (1.5%), and amphetamines (0.5%). The overall prevalence of alcohol and drugs was lower than those reported in previous epidemiological studies of the DRUID project. The year 2011 showed the highest prevalence of drug-positive cases (24.1%), while the lowest prevalence was found in 2016 (16.8%), after the update of the Road Traffic Law (RTL) that increased punishments for driving under the influence. A progressive increase in the number of alcohol-positive female drivers was observed from 2011 to 2018, and the highest prevalence was found in the 26-35-year-old age range. Illicit drugs showed the highest overall prevalence in drivers <26 years of age but, if considering single drugs, cocaine and opiates were mostly found in subjects older than 36 years of age. A higher percentage of drug-positive drivers was found on weekend nights for alcohol and on both weekend and weekday nights for drugs. The types of drugs used by drivers did not change during the studied period.

Prevalence of therapeutic drugs in blood of drivers involved in traffic crashes in the area of Bologna, Italy.

INTRODUCTION: Psychoactive medicines, such as benzodiazepines and Z-drugs (BdZ), antidepressants and antipsychotics (AA) and medical opioids (MO), have an impairing effect on driving ability. In previous epidemiological studies performed on impaired and/or injured drivers, not all relevant psychoactive substances were included in the toxicological assessment, and their prevalence may be underestimated. This study aims to assess the prevalence of a large set of psychoactive substances (n=53) in Italian drivers involved in a road traffic crash and in predefined population subgroups. MATERIALS AND METHODS: The toxicological analyses were performed on the whole blood of 1026 drivers involved in a road traffic crash in the area of Bologna, Italy, from January 2017 to March 2018. Analyses were performed using GC-FID (alcohol), GCMS (illicit drugs) and LC/HRMS (psychoactive drugs). The population was divided into subgroups according to gender, age and crash time. Descriptive statistics were used in order to assess differences among sub-groups. RESULTS: The highest prevalence was found for alcohol (17.3%), followed by medicinal drugs (13.6%) and illicit drugs (5.5%). The prevalence of BdZ, AA and MO were 7.3%, 7.2% and 3.1%, respectively. The frequency of BDZ and AA was significantly higher in female drivers and showed higher prevalence at increasing age. The presence of medicinal drugs was significantly higher during the week and in crashes occurring during the day. CONCLUSION: Results for alcohol and illicit drugs partially overlap with those reported in previous European and Italian studies, but the prevalence of BdZ was much higher. We also found a high prevalence of AA, which are rarely investigated in epidemiological studies performed on drivers, but may cause impairment of the ability to drive, especially when taken in combination with alcohol or other drugs. The pattern of medication use differs from that involving drugs of abuse, since it is mainly observed in female subjects and older drivers and does not follow the same weekly trend observed for alcohol and other illicit drugs.

Validation and preliminary application of a GC-MS method for the determination of putrescine and cadaverine in the human brain: a promising technique for PMI estimation.

INTRODUCTION: Among the several techniques proposed for the estimation of the Post Mortem Interval (PMI), the analysis of odorous amines has been applied in the past, with conflicting results. The aims of this study are: (i) to develop and validate a GC-MS method for the determination of putrescine (PUT) and cadaverine (CAD) in the human brain (validation study) and (ii) to study the relation of PUT and CAD concentration in the human brain and the PMI (decomposition study). MATERIALS AND METHODS: Validation study. Analysis has been performed through GC-MS after a liquid-liquid extraction and a single step-derivatization for the identification and quantification of odorous amines in brain cortex samples. The standard protocol used in forensic toxicology, slightly modified for endogenous compounds according to recent guidelines, was used for validation. Decomposition study. Three uninjured human brains were sampled during the autopsy of three fatal traumatic cases. Along a 120-hour period of decomposition under experimental conditions, each brain was sampled along predetermined time intervals. RESULTS: Validation study. Both PUT and CAD validation parameters were within the acceptable values defined by the Scientific Working Group for Forensic Toxicology (SWGTOX), with better selectivity, linearity, accuracy and precision values for PUT. Decomposition study. A significant relationship between PUT and CAD levels and PMI has been demonstrated through statistical analysis with a correlation coefficient of 0.98 for PUT and 0.93 for CAD (p < 0.0001). CONCLUSION: Although further experimental studies on a wider number of samples are necessary, the results of this study suggest a possible role of polyamine levels in brain cortex for the estimation of PMI.

Optimization of cloned enzyme donor immunoassay cut-offs for drugs of abuse in whole blood of drivers involved in road accidents.

INTRODUCTION: Immunoassay (IA) tests should be able to detect low concentrations of illegal drugs when used for the screening of drugs in drivers. False negatives should be avoided, and false positives should be reduced as far as possible. In this study, semi-quantitative results for blood samples containing illicit drugs (cannabinoids, cocaine, amphetamines/methamphetamines, opiates and methadone) obtained with cloned enzyme donor immunoassay (CEDIA), were compared with results of confirmatory analysis performed through gas chromatography-mass spectrometry (GC-MS). Screening cut-off points for each class of drugs were retrospectively optimized. METHODS: Whole blood samples from drivers involved in road accidents in the period from January 2013-December 2017 were analyzed with CEDIA (4200 samples). Confirmatory analyses were performed through (GC-MS) on: (i) all samples with screening concentrations above 1ng/ml for at least one drug (positive screening results); (ii) 800 samples with screening concentration lower than 1ng/ml (negative screening results). Recommended per se limits in relation to driving under the influence of drugs were set as fixed values. Sensitivity, specificity, positive and negative predictive values were evaluated by contingency tables and compared to ROC-analysis in order to obtain ideal screening cut-offs. RESULTS: CEDIA results were available for 4200 blood samples and 1172 positive screening results were found. Among these, 1008 confirmation analysis were obtained through GC-MS. Optimized screening cut-offs obtained through ROC analysis were as follows: 8.0ng/ml for THC; 5.5ng/ml for THC-COOH; 21.1ng/ml for cocaine; 6.9ng/ml for benzoylecgonine; 33.1ng/ml for opiates; 61.6ng/ml for amphetamines; 5.0ng/ml for methadone. Using these cut-offs, sensitivity was above 97% for THC-COOH, cocaine, benzoylecgonine, amphetamines, opiates and methadone, and 92% for THC; specificity was above 90% for cocaine, benzoylecgonine, amphetamines, opiates and methadone, 80% for THC and 89% for THC-COOH; negative predictive value was above 99% for all drugs and metabolites. CONCLUSION: Previous studies have shown that CEDIA tests are useful for preliminary screening of serum and urine. Its implementation in whole blood is of primary importance for the assessment of impaired driving, since the per se limits of many European countries refer to whole blood, and preparation of the serum and/or the collection of urine is not always possible in the hospital emergency department, where blood samples are withdrawn. Our study shows that CEDIA tests on whole blood permit the definition of cut-off values with optimal sensitivity and negative predictive values for all analytes (near to 100%), including very good specificity.

Medico-legal implications of toluene abuse and toxicity. Review of cases along with blood concentrations.

Toluene, a liquid aromatic hydrocarbon, is one of the most widely used industrial solvents, and is present in numerous paints, paint thinners, glues and other industrial and household products. It has become the most abused solvent in the world due to its rapid effects following inhalation. However, the numerous cases of fatal and non-fatal toluene-related intoxication reported in the literature have not yet been collected and discussed in the forensic setting. In this paper we aim to provide a review of the cases of toluene abuse and intoxication and the state of the art of the forensic toxicological analysis of toluene intoxications in the living and in the dead subject, from the early identification to the medico-legal interpretation of the toxicological result. We have identified a total of 45 papers regarding different aspects of toluene abuse, and divided them into three sections, namely sampling, storage and techniques of analysis, assessment in living subjects and post-mortem assessment. This article reports toluene concentrations in blood from 202 living subjects, 23 fatal toluene intoxications and 85 toluene related deaths. Toxicological results are discussed in relation to the clinical presentation (living subjects, including impaired drivers), and the manner of death according to the medical examiner reports (post-mortem examinations). Finally, we discuss the strengths and limitations of the review.

Toxic cyanobacterial blooms in reservoirs under a semiarid mediterranean climate: the magnification of a problem.

Sicilian reservoirs constitute the most important water resources available on the island. During summer 2001, the intense water utilization of Lake Arancio reservoir reduced the water level significantly, which coincided with the formation of intense blooms formed by the microcystin (MC)-producing cyanobacterium Microcystis aeruginosa. During summer 2003, Lake Arancio was continuously filled and the vertical stratification of the water column was maintained resulting in five to sixfold lower cell numbers of M. aeruginosa. For both years, a significant relationship between MC net production and Microcysytis cell growth was observed, implying that Microcystis cell numbers can be used to infer MC concentrations in water. Unexpectedly, dense blooms of the MC-producing cyanobacterium Planktothrix rubescens occurred during winter 2005/2006 in the reservoirs Lake Pozzillo, Prizzi, Nicoletti, and Garcia but have not been reported earlier. In this season, MC concentrations higher than those recorded in summer were measured, implying that monitoring of Mediterranean drinking water reservoirs needs to be intensified during winter, a season usually considered to be less prone to the formation of cyanobacterial blooms.