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Introduction The estimation of post-mortem interval (PMI) remains a major challenge in forensic science. Most of the proposed approaches lack the reliability required to meet the rigorous forensic standards. Objectives We applied 1H NMR metabolomics to estimate PMI on ovine vitreous humour comparing the results with the actual scientific gold standard, namely vitreous potassium concentrations. Methods Vitreous humour samples were collected in a time frame ranging from 6 to 86 h after death. Experiments were performed by using 1H NMR metabolomics and ion capillary analysis. Data were submitted to multivariate statistical data analysis. Results A multivariate calibration model was built to estimate PMI based on 47 vitreous humour samples. The model was validated with an independent test set of 24 samples, obtaining a prediction error on the entire range of 6.9 h for PMI < 24 h, 7.4 h for PMI between 24 and 48 h, and 10.3 h for PMI > 48 h. Time-related modifications of the 1H NMR vitreous metabolomic profile could predict PMI better than potassium up to 48 h after death, whilst a combination of the two is better than the single approach for higher PMI estimation. Conclusion The present study, although in a proof-of-concept animal model, shows that vitreous metabolomics can be a powerful tool to predict PMI providing a more accurate estimation compared to the widely studied approach based on vitreous potassium concentrations.

Adherence to therapy is the key to a successful therapeutic intervention, especially in cardiovascular diseases in which a lack of adherence may have serious consequences in terms morbidity and/or mortality. In this context, hair analysis can be an excellent tool to monitor adherence to therapy. Indeed, drugs present in blood are incorporated into the hair matrix, where drugs and metabolites can stay unaltered for a long time protected from metabolism and degradation. In the present study, a simple, specific, and sensitive ultra-high performance liquid-chromatography-tandem mass spectrometry (UHPLC-MS/MS) method set up to determine in human hair seven beta-blockers (viz., metoprolol, sotalol, labetalol, atenolol, nebivolol, bisoprolol, and nadolol) and two calcium-channel blockers (lercanidipine and amlodipine), which are widely prescribed to treat medium-to-severe hypertensive conditions. The optimized method was successfully validated in terms of accuracy, repeatability, reproducibility, matrix effect and extraction recovery. Moreover, the applicability of the method was evaluated by analyzing 34 real samples of hair obtained from patients under long-term therapy with calcium channel blockers and beta-blockers.

The magnitude of the diagnostic benefit conferred by performing histopathological examinations after medico-legal/forensic autopsies remains debatable. We have tried to address this issue by reviewing a series of histopathology referrals concerning medico-legal autopsies in real-world routine practice. We present an audit of the consultations provided to forensics by clinical pathologists at our institute between 2015 and 2018. Over this period, 493 post-mortem examinations were performed by forensic pathologists. Of these cases, 52 (11%) were referred for histopathology. Gross assessment was requested in 22/52 (42%) cases. Histopathology examination was performed on single organs in 15/52 (29%) cases, primarily on the lung and heart, whereas parenchymatous multi-organ analysis was carried out in 14/52 (27%) cases. Bone-marrow sampling was studied in 4/52 (8%) cases. Immunohistochemistry was needed in 16/52 (31%) cases, special stains in 9/52 (21%) cases and molecular analysis in 4/52 (8%) cases. Focusing on technical processes, standard methodology on pre-analytical procedures was changed in 10/52 (19%) cases in order to answer specific diagnostic questions. We showed that although most of the time the diagnosis is clear by the end of dissection on the basis of the macroscopic findings, histopathology can provide, modify or confirm the cause of death in many medico-legal/forensic cases. Therefore, it is desirable that forensic pathologists and clinical pathologists establish robust working relationships in a cooperative environment. We conclude that it is important to implement guidelines based on real-world routine practice in order to identify cases where histopathology can provide useful contributions, which in our experience applied to 11% of forensic cases.

According to the World Health Organization, as many as 25% of traffic accidents are linked to alcohol abuse. This study describes the results of a nine-year study performed on injured drivers ( N = 12,806) in the Verona area of Northern Italy. Blood samples were mandatorily collected on injured drivers who were admitted to the Emergency Health Care Unit of Verona Hospital between 2009 and 2017, after they had been involved in a traffic accident. Blood alcohol concentration (BAC) determination was then undertaken using a validated head space–gas chromatography–flame ionisation detector (HS-GC-FID) method. We found that 21% of drivers tested positive for alcohol (BAC ≥0.01 g/L), while 16.8% presented with BAC levels above the Italian legal limit (>0.5 g/L). Of those who had positive BACs, about 50% presented with very high BAC levels (>1.5 g/L). Daily time distribution analyses, involving 2031 alcohol-positive drivers, showed a surge between 18:00 hours and 06:00 hours (74.3%), with a specific rise during the weekend (58.9%). The percentage of alcohol-related road accidents was 20.6%, which is lower than results reported in other international studies performed over the last 20 years. However, evidence that around 50% of the positive subjects showed a BAC >1.5 g/L confirms the correlation between BAC and accident risk, which becomes even more significant at progressively increasing levels of BAC. The study highlights the need to implement further strategies to both prevent and deter the use of alcohol while driving.

Headspace gas chromatography with a flame ionization detector (HS-GC–FID) is a well-established approach for determining blood alcohol concentration, including in cadaveric specimens. Although the integrity of blood specimens can be adequately guaranteed after the sampling, the quantification of ethanol in cadaveric blood can be affected by postmortem fermentative phenomena occurring between the time since death and the sampling of biofluids. The vitreous humor is less affected by putrefactive phenomena allowing compound determination and its use as an alternative biological matrix. The present work aimed to develop and validate a method using the salting-out effect and based on HS-GC–FID for the determination of ethanol in the vitreous humor. The reported analytical method is based on a simple vitreous humor pre-treatment consisting of a dilution (1:9) with a solution of 2.5 mol/L K2CO3 and 0.0012 mol/L tert-butanol (internal standard). After 1 min of incubation, part of the specimen evaporated in the headspace (2,000 µL) is injected into the chromatographic system and analyzed in isothermal mode (40°C), with a chromatographic time of 1.6 min. The method was validated in terms of selectivity, the lowest limit of detection, intraday and total imprecision, and trueness (bias). The determination of ethanol in the vitreous humor and blood was carried out in 75 cases. The correlation between the two matrices was confirmed in 61 cases. However, 14 vitreous humor specimens showed lower ethanol concentrations, and in the related blood specimens, it was possible to identify the signal of n-propanol, a typical product of postmortem microbial fermentation, that justifies the excess of ethanol in the blood specimens.

Cardiac glycosides digoxin and digitoxin are used in therapy for the treatment of congestive heart failure. Moreover, these compounds can be responsible for intoxication cases caused by fortuitous ingestion of leaves of Digitalis. Due to the narrow therapeutic range of these drugs, therapeutic drug monitoring is recommended in the clinical practice. In this context, immunoassays-based methods are generally employed but digoxin- and digitoxin-like compounds can interfere with the analysis. The aim of this study was to develop and validate an original UPLC-MS/MS method for the determination of digoxin and digitoxin in plasma. The method shows adequate sensitivity and selectivity with acceptable matrix effects and very good linearity, accuracy, precision, and recovery. A simple liquid-liquid extraction procedure was used for sample clean-up. The method was applied for the analysis of n = 220 plasma samples collected in two different clinical chemistry laboratories and previously tested by the same immunoassay. The statistical comparison showed a relevant negative bias of the UPLC-MS/MS method versus the immunoassay. These results are consistent with an immunoassay overestimation of digoxin plasmatic levels due to cross-reaction events with endogenous digoxin-like substances.

It is well known that traffic injuries still represent one of the main causes of death and that high blood alcohol concentrations while driving significantly increase the occurrence of accidents. However, only limited literature on the correlation between chronic alcohol abuse and accident risk is available. The aim of the present study was to investigate the hypothesis of an association between elevated concentrations of carbohydrate deficient transferrin (CDT) and the occurrence of alcohol-related traffic accidents.The analytical determinations of BAC and CDT were performed following certified methods in HS-GC-FID and HPLC, respectively. For BAC, 0.50 g/L was used as cut-off, whereas 2.0% was used for CDT, according to the standardisation proposed by IFCC. A total of 929 drivers, tested for BAC at the time of hospital admission after a traffic accident, were classified into two groups: InjDr 1 (BAC ≤ 0.50 g/L) and InjDr 2 (BAC0.50 g/L); all drivers were also tested for CDT.InjDr 1 included 674 individuals, only 2.5% showing a CDT above the cutoff, whereas InjDr 2 group consisted of 255 subjects, 28.6% testing positive for CDT (Odds Ratio 15.5). When subdividing the InjDr group into increasing classes of CDT, a steady increase in the percentage of BAC-positive drivers was appreciated. Moreover, average BAC was found to parallel each class of CDT.The reported data strongly support the use of CDT as a biomarker of increased risk of alcohol-related traffic accidents in the procedures of re-granting of the driving license upon confiscation for "drink driving".

The strong psychoactive effects of synthetic cannabinoids raise the need for the deeper studying of their neurometabolic effects. The pharmacokinetic properties of 5F-APINAC and its influence on metabolomics profiles associated with neurotransmission were investigated in rabbit plasma. Twelve rabbits divided into three groups received 1-mL 5F-APINAC at 0.1, 1 and 2 mg/kg. The intervention groups were compared with the controls. Sampling was performed at nine time points (0–24 h). Ultra-high-performance liquid chromatography–tandem mass spectrometry was used. The pharmacokinetics were dose-dependent (higher curve at a higher dose) with a rapid biotransformation, followed by gradual elimination within 24 h. The tryptophan concentrations abruptly decreased (p &lt, 0.05) in all tested groups, returning to the basal levels after 6 h. 5-hydroxylindole acetic acid increased (p &lt, 0.05) in the controls, but this trend was absent in the treated groups. The aspartic acid concentrations were elevated (p &lt, 0.001) in the treated groups. L-kynurenine was elevated (p &lt, 0.01) in the intervention groups receiving 1 mg/kg to 2 mg/kg. Dose-dependent elevations (p &lt, 0.01) were found for kynurenic acid, xanthurenic acid and quinolinic acid (p &lt, 0.01), whereas the anthranilic acid trends were decreased (p &lt, 0.01). The indole-3-propionic acid and indole-3-carboxaldehyde trends were elevated (p &lt, 0.05), whereas the indole-3-lactic acid trajectories were decreased (p &lt, 0.01) in the intervention groups. 5F-APINAC administration had a rapid biotransformation and gradual elimination. The metabolites related to the kynurenine and serotonergic system/serotonin pathways, aspartic acid innervation system and microbial tryptophan catabolism were altered.

Asialo-human transferrin (asialo-hTf) is a glycoform of the human serum protein transferrin characterized by the lack of the sialic acid (SA) terminal unit. It is known that glycosylation micro-heterogeneity and the presence of SA are strongly involved in protein functioning and pathophysiological activities. Some hTf glycoforms are valuable biomarkers for the detection of both genetic defects of glycosylation and/or sialoform distribution changes. The detection of the carbohydrate deficient transferrin (CDT) glycoforms is currently a widely employed method for the diagnosis of chronic alcohol abuse. The physiological significance of asialo-hTf is still unclear, despite its important biological implications. The current knowledge suggests that asialo-hTf may be involved in regulation of iron transport and release at the hepatic level, which, consequently, could strongly be affected by alcohol consumption. For these reasons, a deeper understanding of asialo-hTf structure and its physiological role is required, and an improved method of its analysis would favor the detection of both chronic abuse and other habits of alcohol intake and/or misuse. Thus, suitable analytical methods possessing higher sensitivity and specificity in comparison with the currently available techniques are certainly recommended. The present review summarizes the studies on asialo-hTf structure, roles, and detection techniques mainly in relation to its possible use as a potentially additional useful biomarker of alcohol abuse, and underlines its prospective value as a forensic and diagnostic tool.

The increase of the carbohydrate-deficient transferrin (CDT) as results of an heavy intake of alcohol for at least two weeks, is a well-known biochemical modification since the middle '70s. Notwithstanding the first commercial kit for the diagnosis of chronic alcohol abuse based on this biomarker was commercially accessible already thirty years ago, only expensive analytical methods are currently available for its determination. The present paper shows a new approach intrinsically sensitive and specific, based on a specific derivatization of transferrin, and not requiring sophisticated instrumentation.The proposed procedure is based on a selective chelation of terbium (III) by transferrin followed by detection using an characteristic Fluorescence Resonance Transfer Energy (FRET) phenomenon (ex 298 nm - em 550 nm).The proposed procedure showed a limit of detection of 2.5 pmol/mL and a reproducibility intra-day and inter-days15% and 20%, respectively. The results obtained analyzing 40 serum samples using the developed method, were compared with those obtained with HPLC-Vis and an RConsidering its main features (low-cost, ease of operation, minimum need of instrumentation) the present method is suitable for application in screening contexts and in non-strictly regulated environments (e.g. clinical diagnosis) as well as in developing countries or remote areas.

A simple and low cost analytical device is described for the determination of ammonium in the vitreous humor suitable for inferring the post mortem interval in forensic cases. The device is based on ammonia formation from ammonium ion by means of NaOH addition to the vitreous humor sample and its detection with a pH chemical indicator in the gas phase above the vitreous humor sample. From the gas phase, ammonia diffuses through a polymeric membrane and it is trapped and detected with a droplet of pH indicator thymol blue. The color change of the droplet is measured using a smartphone camera. Under optimal conditions, the device showed a limit of detection of 0.2mM, with between days precision of ≤ 15% expressed as relative standard deviation, and an accuracy between days from 88.3% to 114.5%. This homemade gas diffusion analytical device was successfully used for the determination of ammonia in vitreous humor samples from forensic autopsies. The results obtained with the proposed method, although for a limited number, showed a close correlation with the data obtained with an instrumental analysis based on capillary electrophoresis. Moreover a significant correlation was also found between the results of the present method and the time elapsed since death by a simple evaluation of the color intensity. In conclusion, this preliminary study showed that the proposed device, after adequate validation, could be a promising tool for a presumptive estimation of the time since death directly at the crime scene.

In the context of fitness certification to hold the driving license, GGT and CDT have been used, sometimes in combination (γ-CDT), to exclude chronic alcohol abuse. The present study was carried out with the aim of comparing the power of these biomarkers as tools for the objective screening of subjects at high risk of alcohol-associated traffic injuries.288 male drivers admitted to hospital after traffic accidents were examined by determination of GGT, CDT and BAC. The degree of association of GGT, CDT and γ-CDT with BAC was analysed using non-parametric statistics.Partitioning the cases using the cut-off concentrations of 0.5 g/L for BAC (the legal limit adopted in most European countries), 55 U/L for GGT and 1.9% for CDT, a highly significant difference was found between the frequency of elevated GGT or CDT in cases where BAC was within the legal limits and those with elevated BAC values (Fisher's exact test: p 0.001). However, the calculation of the odds ratio showed a much higher increase for CDT (28 times) than for GGT (6 times) in those drivers with a BAC above the Italian legal limit in comparison with those showing a BAC within the cut-off; conversely, γ-CDT does not provide any significant advantage vs. CDT alone.Both GGT and CDT provide objective evidence of an association with the occurrence of alcohol-related severe traffic accidents, but CDT shows superior association with these events. Therefore, CDT, notwithstanding higher costs, should be preferred in a forensic/certification context.

In recent years, a significant increase of reports about suicidal cases due to intentional sodium nitrite intake has been described. In the forensic pathology context, the strategy to approach intoxication cases by sodium nitrite, without any preliminary information or hint, is not straightforward. Indeed, in a number of cases the lack of crime scene data and/or specific pathological signs makes difficult the identification of nitrite poisoning. Moreover, the analytical determination of nitrite in blood is challenging, due to its rapid oxidization to nitrate by hemoglobin. Although several methods have been proposed for the clinical analysis of nitrate and/or nitrite in biological samples, none of these is specifically focused on the determination of these ions in cadaveric samples. Consequently, the diagnosis of nitrite fatal intoxication is still based on methemoglobin analysis. The present paper reports the optimization and validation of an analytical method of capillary ion analysis (CIA) with UV detection, for the determination of nitrite and nitrate in biological fluids and its application to two authentic cases of death by nitrite intake. The analyses were carried out in a bare fused-silica capillary (75 µm inner diameter) using 100 mM sodium tetraborate (pH 9.24) as background electrolyte and applying a voltage of − 15 kV between the capillary ends. The detection was obtained by direct UV absorption recorded at 214 nm wavelength. Bromide was used as the internal standard. Linearity was established in the range of 0.25–5 mmol/L). Reproducibility (intraday and day-to-day) was characterized by relative standard deviations (RSDs) 14.7% for peak areas. The method was applied to the determination of nitrite and nitrate in two real forensic cases, where high concentrations of nitrate were found in cadaveric blood samples (6.5 and 4.4 mmol/L, respectively). Nitrite was found only in trace amounts, due to the instability of this ion in cadaveric blood where it is oxidized to nitrate. The present method represents a new tool for the direct and rapid determination of nitrite and nitrate in cases of forensic interest, and thus offers a diagnostic tool more sensitive and precise than the need methemoglobin analysis.

In the present work, dispersive liquid-liquid microextraction (DLLME) was used to extract six synthetic cannabinoids (JWH-018, JWH-019, JWH-073, JWH-200, or WIN 55,225, JWH-250, and AM-694) from oral fluids. A rapid baseline separation of the analytes was achieved on a bidentate octadecyl silica hydride phase (Cogent Bidentate C18; 4.6 mm × 250 mm, 4 μm) maintained at 37 °C, by eluting in isocratic conditions (water:acetonitrile (25:75, V/V)). Detection was performed using positive electrospray ionization–tandem mass spectrometry. The parameters affecting DLLME (pH and ionic strength of the aqueous phase, type and volume of the extractant and dispersive solvent, vortex and centrifugation time) were optimized for maximizing yields. In particular, using 0.5 mL of oral fluid, acetonitrile (1 mL), was identified as the best option, both as a solvent to precipitate proteins and as a dispersing solvent in the DLLME procedure. To select an extraction solvent, a low transition temperature mixture (LTTM; composed of sesamol and chlorine chloride with a molar ratio of 1:3) and dichloromethane were compared; the latter (100 μL) was proved to be a better extractant, with recoveries ranging from 73% to 101 % by vortexing for 2 min. The method was validated according to the guidelines of Food and Drug Administration bioanalytical methods: intra-day and inter-day precisions ranged between 4 % and 18 % depending on the spike level and analyte; limits of detection spanned from 2 to 18 ng/mL; matrix-matched calibration curves were characterized by determination coefficients greater than 0.9914. Finally, the extraction procedure was compared with previous methods and with innovative techniques, presenting superior reliability, rapidity, simplicity, inexpensiveness, and efficiency., Graphical abstract Image 1, Highlights • A DLLME procedure was optimized to treat oral fluid and isolate synthetic cannabinoids. • Acetonitrile was used both as a protein precipitation agent and a dispersing solvent. • Dichloromethane allowed recoveries exceeding 70%. • Rapid baseline separation of analytes was achieved on a bidentate C18 column. • Rapidity, simplicity, efficiency, and high throughput are the main advantages of this procedure.

Estimation of the post-mortem interval (PMI) remains a matter of concern in the forensic scenario. Traditional and novel approaches are not yet able to fully address this issue, which relies on complex biological phenomena triggered by death. For this purpose, eye compartments may be chosen for experimental studies because they are more resistant to post-mortem modifications. Vitreous humour, in particular, has been extensively investigated, with potassium concentration ([K+]) being the marker that is better correlated with PMI estimation. Recently, a 1H nuclear magnetic resonance (NMR) metabolomic approach based on aqueous humour (AH) from an animal model was proposed for PMI estimation, resulting in a robust and validated regression model. Here we studied the variation in [K+] in the same experimental setup. [K+] was determined through capillary ion analysis (CIA) and a regression analysis was performed. Moreover, it was investigated whether the PMI information related to potassium could improve the metabolome predictive power in estimating the PMI. Interestingly, we found that a part of the metabolomic profile is able to explain most of the information carried by potassium, suggesting that the rise in both potassium and metabolite concentrations relies on a similar biological mechanism. In the first 24-h PMI window, the AH metabolomic profile shows greater predictive power than [K+] behaviour, suggesting its potential use as an additional tool for estimating the time since death.

The continuous introduction in the market of new psychoactive drugs (NPS) represents a well-known international emergency. Indeed, the European Monitoring Centre for Drugs and Drug Addiction and the United Nations Office on Drugs and Crime are paying great attention to the spread of NPS. In addition to the traditional analytical approaches based on GC-MS and HPLC-MS, also CE coupled with MS has proved to be a precious tool for the toxicological screening of biosamples. On these grounds, the aim of the present work was to test the application of CE-HRMS as a new screening tool for the rapid detection of these novel drugs in urine. Separations were performed in an uncoated fused-silica capillary with id of 75 μm with a total length of 100 cm, by applying a constant voltage of 15 kV. The QTOF-MS was implemented with an electrospray ion source operating in positive ionization full scan mode in the range of 100-1000 m/z. Under these conditions, different NPS has been tested, including eight cathinones, five phenethylamine, and seven tryptamines. The method was validated after optimization of the following analytical parameters: BGE composition and pH, separation voltage, sheath liquid composition, and flow rate and ESI source settings. The applicability of the method was successfully tested by analyzing a series of real urine samples obtained from drug users.

Ranolazine is a selective inhibitor of the late inward sodium current, approved for the treatment of chronic angina. Here, a case of a possibly suicidal death due to acute ranolazine overdosing is reported. A 41-year-old woman was found unconscious by her son and was urgently admitted to the intensive care unit. She had ingested an unknown amount of ranolazine tablets. Seventeen hours after admission, the patient died. An autopsy was performed 4 days postmortem. A routine screening analysis for drugs of abuse and medicinal drugs performed by liquid chromatography (LC)–ion trap mass spectrometry (MS) on autopsy samples of biological fluids did not detect any relevant presence of toxicologically relevant compounds, but ranolazine. A quantitative analysis was then carried out by liquid chromatography--triple quadrupole (LC–QqQ) MS in order to quantify ranolazine and its major metabolite o-desmethylranolazine in biological fluids and organs. Ranolazine concentrations in biological fluids were as follows: cardiac blood, 19.5 μg/mL; femoral blood, 12.3 μg/mL; bile, 0.87 μg/mL and vitreous humor, 15.4 μg/mL. For O-desmethylranolazine, the concentrations in cardiac blood, femoral blood, bile and vitreous humor were 10.7, 9.6, 11,103 and 11.4 μg/mL, respectively. The cause of death was attributed to ranolazine overdosing. To the best of our knowledge, this is the first report of a fatality associated with ranolazine, in which the postmortem distribution of ranolazine and its metabolite has been quantitatively assessed. The present study can therefore provide useful information for interpretation of the causes and mechanisms of death in ranolazine-associated fatalities.

Diazepam is a well-known psychoactive drug widely used worldwide for the treatment of anxiety, seizures, alcohol withdrawal syndrome, muscle spasms, sleeplessness, agitation, and pre/post-operative sedation. It is part of the benzodiazepine family, substances known to primarily act by binding and enhancing gamma-aminobutyric acid (GABAShort-term (2.5 h) and medium-term (96 h) exposures to diazepam were performed at drug concentrations of 0.8, 1.6, 16, and 160 μg/L. Intervention groups were compared with a vehicle control group. Each group consisted of 20 zebrafish eggs/larvae. Metabolites related with neurotransmission were determined by ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS).Thirty-six compounds were quantified. Significantly increased tryptophan and serotonin concentrations were found in the intervention groups receiving higher doses of diazepam in 2.5 h exposure (p 0.05 control versus intervention groups). Tyrosine concentrations were higher (p 0.05) at higher concentrations in 2.5 h exposure, but lower (p 0.05) at higher concentrations in 96 h exposure. Both phenylalanine and aspartic acid concentrations were higher (p 0.05) at higher doses in 2.5 h and 96 h exposure.Short- and medium-term exposures to diazepam induce dose- and time-dependent metabolomic alterations associated with the serotonergic, dopaminergic/adrenergic, and aspartic acid neurotransmitter systems in zebrafish.

Post-mortem changes of ions in the body fluids have been proposed as an objective tool for inferring the time of death. In particular, the post-mortem increase of potassium concentrations in the vitreous humour has gained great attention in the literature. On the other hand, ammonium, another ion released in post-mortem processes, has received much less attention, potentially due to unresolved analytical issues using current clinical chemistry methods. This paper presents an application of a new analytical approach based on capillary electrophoresis providing the simultaneous analysis of potassium and ammonium ions in the vitreous humour. In addition, to assess the consistency of the post-mortem increase of ammonium concentrations in the vitreous humour, the determination of this ion in the vitreous humour of the two eyes of the same body at the same post-mortem interval has been verified. Vitreous humour was collected from 33 medico-legal cases where the time of death was known exactly. Prior to analysis, all samples were diluted 1:20 with a 40 μg/mL solution of BaCl2 (internal standard). In the study of the variability of the ammonium concentration between the two eyes, no statistically significant differences were found, supporting the hypothesis of an even post-mortem increase of the ion concentrations in this particular biological fluid. Significant correlations of potassium and ammonium ions with the post-mortem interval were found, with r2 of 0.75 and 0.70, respectively.

The biomarkers can provide objective and relevant information for the clinical and/or forensic diagnosis of alcohol abuse. Many researchers spent efforts not only to find new markers of alcohol abuse, but also to verify in real conditions the specificity and sensitivity limits of the existing biomarkers and to optimize the analytical methodologies for their determination. The great attention dedicated by clinical and forensic researchers is proved by the several hundreds of papers published on this subject, which can be retrieved in the main databases using the key words “alcohol abuse” and “biomarkers.” A general classification of the markers of alcohol abuse includes two sub-groups: “trait markers” and “state markers.” The most typical biomarkers for the diagnosis of alcohol abuse include gamma-glutamyltransferase, mean corpuscular volume, liver enzymes, aspartate-transaminase and alanine-transaminase, and carbohydrate-deficient transferrin (CDT). In the clinical context, CDT in longitudinal studies proved suitable for the identification of relapses after a period of abstinence.

Introduction and aim Carbohydrate Deficient Transferrin (CDT) is one of the most used biomarkers for monitoring alcohol use in pregnancy. However, its effective application in this context is hampered by the demonstrated physiological progressive increase during pregnancy (even in abstinent women) of CDT values, which in the third trimester can reach values close or exceeding the cut-offs usually adopted in clinical and forensic diagnostics. The present work was aimed at the re-assessment of CDT reference values in pregnancy. Materials and methods The CDT analysis was performed by a validated HPLC-UV Vis method on 284 serum samples of women with a physiological pregnancy and on 370 sera of non-pregnant woman from the general population (control group). All the samples were tested also for GGT for excluding alcohol abuse. The statistical analysis was performed using the MedCalc® Statistical Software. Results The re-definition of the specific reference concentrations was carried out according to the Horn and Pesce Robust Method. The resulting CDT upper reference values were 1.45%, 2.01% and 2.05% in the first, second, and third trimester, respectively. Conclusions In order to prevent the development of maternal and fetal prenatal alcohol exposure complications, the use of alcohol biomarkers, including CDT, has been proposed. However, this biomarker, in the monitoring of alcohol use in pregnancy, has so far been applied adopting the same cut-off used for general population without taking into consideration the progressive physiological increase of its value throughout the pregnancy. In the present study, a specific re-assessment of the CDT reference concentrations of each trimester is reported.

The increased diffusion of the so-called novel psychoactive substances (NPS) and their continuous change in structure andconceivably activity has led to the need of a rapid screening method to detect their biological effects as early as possible after their appearance in the market. This problem is very felt in forensic pathology and toxicology, so the preclinical study is fundamental in the approach to clinical and autopsy cases of difficult interpretation intoxication. Zebrafish is a high-throughput suitable model to rapidly hypothesize potential aversive or beneficial effects of novel molecules. In the present study, we measured and compared the behavioral responses to two novel neuroactive drugs, namely APINAC, a new cannabimimetic drug, and methiopropamine (MPA), a methamphetamine-like compound, on zebrafish larvae (ZL) and adult mice. By using an innovative statistical approach (general additive models), it was found that the spontaneous locomotor activity was impaired by the two drugs in both species: the disruption extent varied in a dose-dependent and time-dependent manner. Sensorimotor function was also altered: i) the visual object response was reduced in mice treated with APINAC, whereas it was not after exposure to MPA; ii) the visual placing responses were reduced after treatment with both NPS in mice. Furthermore, the visual motor response detected in ZL showed a reduction after treatment with APINAC during light-dark and dark-light transition. The same pattern was found in the MPA exposed groups only at the dark-light transition, while at the transition from light to dark, the individuals showed an increased response. In conclusion, the present study highlighted the impairment of spontaneous motor and sensorimotor behavior induced by MPA and APINAC administration in both species, thus confirming the usefulness of ZL as a model for a rapid behavioural-based drug screening.

New generations of synthetic cannabinoids (SCs) have recognized strong psychoactive effects, rapidly becoming potent drugs of abuse. Characterization of metabolites associated with SCs is useful for their subsequent detection in biological samples. We aimed to investigate the metabolism of 5F-APINAC, a novel SC, in human liver microsomes (HLMs) (in vitro) and in a rat model (in vivo). For the in vitro study, the standard solution of 5F-APINAC was incubated with HLMs for 1 h at 37 °C. In the in vivo study, rats received 15 mg/kg of 5F-APINAC diluted in ethanol or in dimethyl sulfoxide, while a vehicle control group received placebo. Urine was collected 3, 6, and 24 h after administration. The metabolic characterization was performed using liquid chromatography–ion trap mass spectrometry and liquid chromatography–quadrupole time-of-flight mass spectrometry. In total, 15 metabolites associated with 5F-APINAC were tentatively identified. The metabolites were classified as: 1-adamantol metabolites, oxidative defluorination metabolites, and N-fluoropentylindazole-3-carboxylic acid metabolites. The modifications included ester hydrolysis, mono-, di-, and trihydroxylation of adamantyl ring and N-fluoropentylindazole moiety, oxidation (carbonyl formation) of the N-fluoropentyl side chain, oxidative loss of fluorine, and glucuronidation, as well as combinations thereof. The predominant metabolic reaction was ester hydrolysis in both in vitro and in vivo experiments and formation of M9 (5-fluoropentylindazole-3-carboxylic acid). However, the most recommendable metabolites for proving 5F-APINAC consumption in urine were M4.1, M7, and M13. No metabolites were detected in rat’s urine 24 h after drug administration. The discovered metabolites are proposed to be incorporated into routine screening analytical methods as the urine markers of 5F-APINAC consumption. This is the first report to demonstrate the metabolism of 5F-APINAC to our knowledge.

The toxicological approach for monitoring Driving Under Influence of Drugs (DUID) requires analytical techniques with a broad spectrum of identification coupled to a high analytical sensitivity. In this context immunological methods are generally used, while GC or LC-MS are applied for the confirmation step. A different approach for drug screening is represented by the Toxtyper™ instrumentation, an LC-MS platform equipped with a high-speed ion trap mass analyzer, provided with ready-to-use protocols and a database of as many as 4500 therapeutic, toxic/illicit drugs and metabolites. The aim of the present work was to verify its performances in real conditions of drug screening of human serum in the context of DUID. To test and compare its analytical performances, four pooled serum samples were fortified with a selected panel of 47 drugs and metabolites. The agreement between the results from the ToxtyperTM and from the confirmatory techniques currently in use at the University of Verona (GC and LC-MS) was investigated by analyzing 90 real samples chosen from those routinely analyzed. The present study highlights the suitability of the ToxtyperTM for drug screening in serum with a sensitivity compatible with the needs of the DUID for all the tested compounds, with the only exception of cannabinoids.

The polysialylated isoform of the neural cell adhesion molecule (PSA-NCAM) has been shown to be a key player in neuroplastic changes and is expressed in various disorders. We investigated the PSA-NCAM expression on brain cortical tissue in a cohort of drug-related deaths. Brains from 25 drug abusers and 10 control subjects were removed at autopsy, and 2 samples of the right parietal lobe of each case were obtained. The polysialylated isoform of NCAM was evaluated on formalin-fixed and paraffin-embedded tissues. Eleven patients were polydrug abusers; 14 used a single substance. The mechanisms of death were acute respiratory failure (n = 19), cardiorespiratory failure (n = 4), acute heart failure (n = 1), and brain injury (n = 1). Toxicological analyses of blood were available for all cases, and urine and bile analyses for 19 of 25 cases. The polysialylated isoform of NCAM immunoexpression in the neuronal soma and dendritic spines was observed in 18 (72%) of 25 drug abusers and in 2 (20%) of 10 control subjects. Drug abusers were statistically more positive for PSA-NCAM than control subjects (P = 0.0082). The expression of PSA-NCAM in the parietal cortex could be an indicator of brain damage due to drug abuse, and its availability could allow the forensic pathologists to develop rapid and low-cost additional or alternative method to improve detection of drug-related deaths.

Accurate dating of bone fractures can be crucial in the context of forensic trauma analysis since it may provide essential information for the corroboration or contradiction of statements by victims or suspects in cases of domestic abuse or torture. The different stages of bone healing have been well described in the existing literature, and some previous studies attempted to define related timelines particularly in the paediatric population. However, the bone healing process can be very variable and despite the importance of the topic in the forensic field, so far little is known about the radiological appearance of bone fractures at different healing stages, and how this correlates with time, especially in an adult population. The aim of this retrospective observational study was to describe the temporal aspect of pre-defined healing stages as they appear on radiographs of tubular bone fractures in adults, and explore the effect of potential cofounding variables. A dynamic nomogram was developed as a user-friendly tool to be eventually applied in clinical or medico-legal settings. This study showed that the posttraumatic time interval (PTTI) increased progressively with the pre-defined healing stages. However, confounding factors, such as patients' age, sex, and location of the fracture need to be accounted for in the final estimation model. Further studies are needed to explore more potentially confounding variables to refine the presented outcomes. Better knowledge of the effect of different confounding variables in the dating of fracture healing will contribute to greater accuracy of PTTI estimation of bone fractures in adults.

Transferrin is a glycoprotein containing two bi- or tri-antennary carbohydrate chains ending with sialic acid. Its glycosylation is reduced in chronic alcohol abuse and in inborn glycosylation pathologies, where the carbohydrate-deficient fraction of the protein (CDT) increases significantly. The current methods require a gradient chromatographic separation and time-consuming sample preparation. In comparison, the proposed approach uses a novel flow-modulated liquid chromatography technique (fmLC) and a highly selective and sensitive fluorescence derivatization reaction with terbium ion. A fmLC-FLD method using isocratic anion exchange separation was optimized and validated to resolve disialo-transferrin and trisialo-transferrin from other transferrin glycoforms. Detection took place by recording fluorescence at 550 nm wavelength (excitation at 298 nm). The chromatographic separation needed 5 min, allowing seriate injection every 7.5 min. The method was validated according to the current guidelines of analytical chemistry showing adequate accuracy and precision for the quantitative determination of CDT. The proposed method proved also to be suitable to analyse haemolyzed sera which, because of interference by haemoglobin, fail the standard HPLC-Vis analysis. The method was tested in parallel with HPLC-Vis on 131 sera showing an excellent correlation of results proved by a correlation coefficient of 0.995 (Pearson’s r). The proposed approach proved much simpler than the current methods and cheaper in terms of instrumental costs offering a ground-breaking analytical tool that could likely make available the characterization of CDT outside specialized laboratories, such as in occupational medicine centres, doctor’s offices, small laboratories, alcohol rehabilitation centres, and in developing countries.

The use of paper-based microfluidics devices (μPADs) for designing rapid, easy, and on-site devices has undergone a dramatic increase in the last few years and the development of applications in the field of forensic science has had concomitant advances. The use of μPADs at the crime scene is finalized not only to obtain a rapid response but also to have crucial information on evidence collection. Several authors have proposed μPADs as alternative approaches to existing tests. These devices are designed to be used on-site without, or minimal, power sources. Furthermore, the technology provides prompt-readouts using a small sample volume, preserving important evidence for instrumental analysis. This review aims to provide a critical evaluation of developed μPADs. The document includes a brief overview on fabrication methods and on detection strategies, and it assesses advantages and limitations of applications related to forensic matters. A final section is devoted to future developments.

The recent appearance of APINAC (AKB-57, ACBL(N)-018, adamantan-1-yl 1-pentyl-1H-indazole-3-carboxylate) in the market of the so-called novel psychoactive substances resulted in the need of defining its characteristics and searching its metabolites for subsequent detection in biological samples. The structure of the APINAC molecule has great similarity to the molecules of other synthetic cannabinoids. Here we report on the in vivo metabolism of APINAC using rats as an experimental model. Rat urine samples were analyzed by using gas chromatography–mass spectrometry and liquid chromatography–high resolution mass spectrometry. Data were acquired via time-of-flight mass scan, followed by Auto MS and triggered product ion scans. The predominant metabolic pathway for APINAC was ester hydrolysis yielding a wide variety of N-pentylindazole-3-carboxylic acid metabolites and 1-adamantanol metabolites. Ten metabolites for APINAC were identified, with the majority generated by hydroxylation, carbonylation, and carboxylation with or without glucuronidation. Therefore, in vivo metabolic profiles in rats were generated for APINAC. N-Pentylindazole-3-carboxylic acid, hydroxylated N-pentylindazole-3-carboxylic acid, and 1-adamantanol are likely the best targets to incorporate into analytical screening methods for drugs analysis. The presented mass spectra and retention time data may be useful for detection of these compounds in human urine.

Molecular imprinted poly(acrylamido)-derivative nanogels have shown their selectivity to bind the protein human serum transferrin (HTR) and also showed their capability for instantaneous solvent-induced modification upon the addition of acetonitrile. Integrated to matrix-assisted laser desorption/ionization time-of-flight mass analysis the HTR-imprinted solvent-responsive nanogels permitted the determination of HTR straight from serum and offered novel perspectives in targeted protein analysis.

INTRODUCTION: This study aimed to establish whether an alcoholic antiseptic, wiped or not before venipuncture, may jeopardize alcohol testing with a commercial enzymatic assay and a reference head-space gas chromatography (GC) technique. MATERIALS AND METHODS: Venous blood was collected from 23 healthy volunteers, with two sequential procedures. In the first blood collection, 2 mL of alcoholic antiseptic (0.5% chlorhexidine, 70% ethanol) were place on a gauge pad, the venipuncture site of right arm was cleaned but the antiseptic was not let to dry before phlebotomy. In the second blood collection, 2 mL of the same alcoholic antiseptic were placed on another gauge pad, the venipuncture site of left harm was cleaned and the antiseptic was accurately cleansed before phlebotomy. Ethanol was measured with a reference GC technique in whole blood and EDTA plasma, and a commercial enzymatic assay in EDTA plasma. RESULTS: No subject complained about feeling a particular itchy sensation when the alcohol was not wiped before puncturing the vein. The concentration of alcohol in all EDTA plasma samples was always lower than the limit of detection of the enzymatic assay (i.e., 2.2 mmol/L ; 0.1 g/L). Similarly, alcohol concentration was also undetectable using a reference GC technique (i.e., < 0.22 mmol/L ; 0.01 g/L) in EDTA plasma and whole blood. CONCLUSION: It seems reasonable to conclude that using ethanol-containing antiseptics before venipuncture may not be causes of spurious or false positive results of alcohol measurement at least when ideal venipunctures can be performed.

Forensic inquests, particularly, in assessing time since death currently recognize the importance of the analysis of vitreous humour (VH) biomarkers. Present research, studies, and validates the determination of lactate (La) in VH by CZE with indirect UV detection. The BGE (pH 8.9) consisted of Tris buffer (37 mM) containing 4-methoxybenzoic acid (4 mM) and alkyl-trimethyl-ammonium bromide (1.2 mM). Each VH specimen was diluted with a butyric acid solution (internal standard 0.057 mM) and La and butyrate were separated within 3-5 min (30 kV). The La LOQ and LOD were 4 and 2 mM, respectively. The calibration curve linearity ranged from 4 to 80 mM; intra- and interruns precisions were less than 10% for standard as well as for VH specimen, respectively. To investigate postmortem interval (PMI) and VH lactate level correlation, human VH specimens were collected during autopsy (n = 40) and stored at -20°C until assay. La levels ranged from 16 to 42 mM; PMI values ranged from 10 to 141 h. La (mM) and PMI (h) correlation was statistically significant (r2 = 0.527; p < 0.05). In conclusion, the present CZE analysis is efficacious to determine VH La as a biomarker for PMI investigation.

Most of the on-site approaches for inferring of the post-mortem interval are still based on observative data from the direct body inspection, whereas, objective and quantitative analyses, such as potassium in the vitreous humor, are require laboratory instrumentation and skilled personnel. The present paper presents a simple and low cost analytical method suitable for use at the crime scene for inferring the time since death. The method uses a microfluidic paper-based device (μPAD) for the determination of ammonium in the vitreous humor (VH) based on the selective interaction between the ammonium and the Nessler's reagent. The color change was measured in terms of “RGB distance” by using a simple and free smartphone application. The optimized device showed a limit of detection of 0.4 mmol L−1, with between days precision less than 9.3% expressed as relative standard deviation, and accuracy between days from 94.5% to 104.5%. The selectivity of the Nessler's reaction was tested towards the main vitreous humor compounds, and no significant interferences were found. This paper-based analytical device was successfully used for the determination of ammonium ion in VH samples from forensic autopsies. The results obtained with the proposed method, although for a limited number of cases (n = 25), showed a close correlation with the data obtained with an instrumental analysis based on capillary electrophoresis. Moreover, in order to make the evaluation of results as simple as possible, a direct correlation between the color intensity, expressed as RGB distance, and the post-mortem interval was studied and a significant correlation was found (R2 > 0.78). In conclusion, the present preliminary study showes that the proposed device could be an additional tool to the traditional methods for a more accurate, although still presumptive, estimation of the time of death directly at the crime scene.