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• Alcohol abuse during pregnancy can cause severe maternal and fetal complications. • Different alcohol biomarkers are used for identifying this condition. • CDT is recognized as a sensitive and specific biomarker of chronic alcohol abuse. • During pregnancy a physiological progressive increase of CDT values occurs. • It hampers the adoption of the usual CDT cut-offs in pregnancy. • The work aimed at reassessing the CDT reference concentrations in pregnancy. 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. 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. 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. 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. [ABSTRACT FROM AUTHOR]

Background: Erythrocyte mean corpuscular volume (MCV) has been used for decades as a biomarker of chronic alcohol abuse and in the treatment of alcohol dependence. More recently, it has also been adopted to investigate the fitness of subjects to hold the driving license to prevent traffic accidents. So far, however, the studies on the association of MCV with an increased risk of alcohol‐associated car accidents are extremely scarce, if not totally absent. To the best of our knowledge, the present work is the first specifically aimed at studying a plausible association between elevated MCV and crash accidents correlated with alcohol abuse. Methods: A total of 6,244 drivers involved in traffic accidents underwent mandatory laboratory analyses including blood alcohol concentration (BAC) determination and MCV analysis. BAC and MCV determinations were performed by headspace gas chromatography and complete blood count, respectively. Results: The chi‐square test evaluating the proportions of subjects with elevated MCVs (>95 fl) yielded a highly significant result (χ2 = 68.0; p < 0.001) in the blood samples where the BAC was above the legal limit (i.e., >0.5 g/l). However, when considering only drivers showing BACs in the range of 0.51 to 1.5 g/l, the frequencies of elevated MCV values are fairly comparable (χ2 = 0.062, p = 0.80). In contrast, limiting the evaluation to BACs > 1.5 g/l, the frequency of elevated MCVs raised to 19.1% (χ2 = 58.9, p value < 0.001 vs. the group with BAC within the legal limits). Conclusions: The present observations show that MCV increases are typically associated with drivers involved in accidents only if driving under severe alcohol intoxication, leading to a preliminary conclusion that, in the context of the certification of the fitness to the driving license, MCV fails to reveal individuals at risk who tend to drive in a condition of low‐to‐moderate alcohol intoxication. [ABSTRACT FROM AUTHOR]

Background Carbohydrate-deficient transferrin ( CDT) is a well-recognized highly specific marker of chronic alcohol abuse. The association of CDT with alcohol-related traffic accidents was evaluated to objectively validate the use of this marker for certifying the physical fitness for driving license regranting after its confiscation for drunk driving. Methods The study was carried out on 468 injured drivers (InjDr), who underwent mandatory blood alcohol concentration ( BAC) and drug analysis in biological fluids. The InjDr group was divided into 2 subgroups on the basis of BAC legal limit adopted in Italy ( BAC ≤ 0.5 g/l: InjDr1; BAC >0.5 g/l: InjDr2). The control group (CntDr) included 236 subjects holding safety-sensitive job positions and undergoing mandatory toxicological analyses. The determination of BAC in blood and CDT in serum were performed using validated analytical methods based on head-space gas chromatography and high-performance liquid chromatography, respectively. Results The evaluation of CDT distribution in the 3 groups (CntDr, InjDr1, InjDr2) showed that CDT distribution in the InjDr1 group was similar to that observed in the CntDr group ( p = 0.159) and different from that observed in the InjDr2 group ( p < 0.001). Partitioning the CDT data of each group into ' CDT positives' and ' CDT negatives' on the basis of the cut off (1.90%), it was possible to calculate the odds of the 3 groups and then the odds ratios. The odds ratio of InjDr1 versus CntDr was 4.56 ( p = 0.158), whereas the odds ratio of InjDr2 versus CntDr was 132 ( p < 0.001). Furthermore, a dose-response effect was found only when comparing InjDr2 with CntDr. Conclusions The data of the present study strongly support the use of the CDT test to evaluate the risk of a subject to be involved in a road accident while driving under the influence of alcohol. [ABSTRACT FROM AUTHOR]

Abstract: Background: The determination of Carbohydrate Deficient Transferrin (CDT) in a forensic context should be based on the use of a screening technique followed, for the “positive samples”, by a confirmatory technique. The aim of this study was to compare the two most used automated screening methods for CDT analysis, immuno-nephelometric assay (INA) and multi-capillary electrophoresis (mCE), with a validated HPLC procedure, used as confirmation test, in order to re-evaluate the cut-off concentrations of the screening methods. Methods: 195 serum samples underwent CDT analysis by using the N Latex CDT direct immuno-nephelometric assay, the multicapillary system Capillarys™ and an anion exchange HPLC method with UV-visible detection at 460nm developed and validated at our laboratories. Statistical analyses were performed by using Bland–Altman plots and ROC curves. Results and discussion: The 95% limits of agreement were ±0.94% when comparing INA and HPLC and ±0.60% when comparing mCE and HPLC. The ROC analysis of both INA and mCE, using HPLC as the reference method, showed that no false negative results were found when the cut-off was fixed to 1.2% for mCE and to 2.3% for INA. Conclusions: The study showed a good agreement among CDT determinations carried out either with mCE or INA or HPLC. However, the usual cut-offs of both mCE (1.3%) and INA (2.5%) should be lowered to minimize false negatives at the screening analysis. [Copyright &y& Elsevier]

Background: 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.Methods: 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.Results: 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.Conclusions: 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. [ABSTRACT FROM AUTHOR]

Abstract: Carbohydrate-deficient transferrin (CDT) is a marker of chronic alcohol abuse, which has recently been introduced to evaluate the physical fitness for obtaining a driving license. The aim of the present study was to evaluate the prevalence of elevated CDT levels in subjects stopped while driving under the influence of alcohol by using a validated method based on capillary electrophoresis. The study was carried out on a group of 40 drunken drivers (group A) and on a control group (n =51) of subjects chosen from the general population (group B). CDT was directly determined by capillary electrophoresis in free solution and UV detection at 200nm. CDT results from both groups were classified as “negative” or “positive” on the basis of the cut-off set at 2.00% (CDT index). The subjects classified as “positive” in group A were 24 (60%), whereas in group B were 2. The subjects classified as “negative” in group A were 16 (40%), whereas in group B was 49 (96.1%). The comparison of the observed percentages, evaluated with the χ 2-test, was highly significant (p <0.001). The present study confirms the high prevalence of chronic alcohol abusers among drunken drivers and the usefulness of CDT as a predictor of the risk of drunk driving. [Copyright &y& Elsevier]

Abstract: In forensic toxicology, hair analysis has become a well established analytical strategy to investigate retrospectively drug abuse histories. In this field, gas chromatography–mass spectrometry and high-performance liquid chromatography–mass spectrometry are currently used, often after preliminary screening with immunoassays. However, on the basis of previous applications to pharmaceutical analysis, capillary zone electrophoresis coupled to ion trap mass spectrometry looks also highly promising. The purpose of the present work was the development of a simple and rapid CZE–MS method for sensitive and quantitative determination of the main drugs of abuse and their metabolites (namely, 6-monoacetylmorphine, morphine, amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethampthetamine (MDMA), benzoylecgonine, ephedrine and cocaine) in human hair. Hair samples (100mg) were washed, cut and incubated overnight in 0.1M HCl at 45°C, then neutralized with NaOH and extracted by a liquid–liquid extraction method. CZE separations were carried out in a 100cm×75μm (I.D.) uncoated fused silica capillary. The separation buffer was composed of 25mM ammonium formate, pH 9.5; the separation voltage was 15kV. Electrokinetic injections were performed at 7kV for 30s under field amplified sample stacking conditions. ESI-ion trap MS detection was performed in the ESI positive ionization mode using the following conditions: capillary voltage 4kV, nebulizer gas (nitrogen) pressure 3psi, source temperature 150°C and drying gas (nitrogen) flow rate 8l/min. A sheath liquid, composed of isopropanol–water (50:50, v/v) with 0.5% formic acid, was delivered at a flow rate of 4μl/min. The ion trap MS operated in a selected ion monitoring mode (SIM) of positive molecular ions for each drug/metabolite. Collision induced fragmentation was also possible. Nalorphine was used as internal standard. Under the described conditions, the separation of all compounds, except amphetamine/methamphetamine, MDA/MDMA and morphine/6-MAM was achieved in 20min, with limits of detection lower than the most severe cut-offs adopted in hair analysis (i.e. 0.1ng/mg). Linearity was assessed within drug concentration ranges from 0.025 to 5ng of each analyte/mg of hair. Analytical precision was fairly acceptable with RSD''s ≤3.06% for migration times and ≤22.47% for areas in real samples, in both intra-day and day-to-day experiments. On these grounds, the described method can be proposed for rapid, selective and accurate toxicological hair analysis for both clinical and forensic purposes. [Copyright &y& Elsevier]

The current application of capillary electrophoresis in forensic toxicology has been critically reviewed with special focus on the areas where this technique has shown real advantages over chromatographic methods. For example, capillary electrophoresis has been most successfully applied to the chiral analysis of some drugs of forensic interest, including amphetamines and their congeners. Another typical application field of capillary electrophoresis is represented by protein analysis. Recently, special interest has been paid to carbohydrate deficient transferrin (CDT), the most important biological marker of chronic alcohol abuse. Other specific applications of capillary electrophoresis of potential forensic toxicological concern are also discussed. The review includes 62 references. [ABSTRACT FROM AUTHOR]

Abstract: The diagnosis of alcohol abuse based on objective data is a necessary requirement in both clinical and forensic environments. Among the different biomarkers of chronic alcohol abuse, carbohydrate-deficient transferrin (CDT) is world wide recognized as the most reliable indicator. However, several problems about the real meaning of CDT and the reliability of its use for the diagnosis of alcohol abuses are still open, as reported by numerous research articles and reviews. The present article presents a critical review of the literature on CDT appeared in the period from 2001 to 2005 (included). The article is organized in the following sections: (1) introduction, (2) definition and structure of human serum CDT, (3) pathomechanisms of the ethanol-induced CDT increase, (4) preanalysis, (5) analysis, (6) data interpretation, (7) review papers, (8) conclusions. As many as 127 papers appeared in the international literature and retrieved by the search engines PubMed and Scopus are quoted. [Copyright &y& Elsevier]

The present work was aimed at the development of a capillary electrophoretic analysis of γ-hydroxybutyric acid (GHB) using electrospray ion trap mass spectrometry to achieve the direct and unequivocal detection of this analyte in human urine. Optimized capillary electrophoretic conditions were: injection, 20s at 0.5psi (1 psi = 6894.76Pa); buffer electrolyte, 12.5mM ammonium formate adjusted to pH 8.35 with diethylamine; fused silica capillary: 100cm × 50μm i.d.; separation voltage, 25kV (forward polarity) + 0.5psi; room temperature. Electrospray and mass spectrometric conditions were: drying gas and nebulizing gas (nitrogen) at flow rate 3l/min, temperature 250°C, nebulizer pressure: 10psi; sheath liquid solution: methanol–water (90:10) containing 0.1% ammonia delivered at 3μl/min; spray voltage 3.5kV. Mass spetrometric detection was carried out in the selected ion monitoring mode of negative molecular ions at 103 m/z for GHB and 115 m/z for maleic acid (I.S.). Under these conditions the baseline separation of GHB and the I.S. was obtained. The selectivity of the analysis allowed for direct injection of unextracted urine, previously diluted 1:4 with water. Linearity was assessed in the GHB concentration range from 80 to 1280μg/ml in urine. Analytical sensitivity (as limit of detection) resulted about 5μg/ml in water and 20μg/ml in original urine. Analytical precision was fairly acceptable with R.S.D. values lower than 5% for migration times and 18% for quantitation in real samples, in both intra day and day-to-day experiments. On these grounds, the developed method can be adopted for rapid identification of acute intoxications from GHB in humans. [Copyright &y& Elsevier]

γ-Hydroxybutyric acid (GHB) is a central nervous system (CNS) depressant and hypnotic which, in recent times, has shown an increasing abuse either as recreational drug (due to its euphoric effects and ability to reduce inhibitions) or as doping agent (enhancer of muscle growth). Analogues of GHB, namely γ-butyrolactone (GBL) and 1,4-butanediol (1,4-BD), share its biological activity and are rapidly converted in vivo into GHB. At present, GHB and analogues are placed in the Schedules of Controlled Substances. Numerous intoxications in GHB abusers have been reported with depressive effects, seizures, coma and possibly death. The purpose of the present work was the development of a rapid analytical method based on capillary zone electrophoresis for the direct determination of GHB in human urine and serum at potentially toxic concentrations. Analytical conditions were as follows. Capillary: length 40 cm (to detector), 75 μm i.d.; buffer: 5.0 mM Na2HPO4, 15 mM sodium barbital adjusted to pH 12 with 1.0 M NaOH; voltage: 25 kV at 23 °C; indirect UV detection at 214 nm; injection by application of 0.5 psi for 5 s. α-Hydroxyisobutyric acid was used as internal standard (IS). Sample pretreatment was limited to 1:8 dilution. Under these conditions, the sensitivity was ∼3.0 μg/ml (signal-to-noise ratio >3). Calibration curves prepared in water, urine and serum were linear over concentration ranges 25–500 μg/ml with R2≥0.998. Analytical precision was fairly good with R.S.D.<0.60% (including intraday and day-to-day tests). Quantitative precision in both intraday and day-to-day experiments was also very satisfactory with R.S.D.≤4.0%. No interferences were found neither from the most common “drugs of abuse” nor from endogenous compounds. In conclusion, capillary electrophoresis can offer a rapid, precise and accurate method for GHB determination of biological fluids, which could be important for screening purposes in clinical and forensic toxicology. [Copyright &y& Elsevier]

Ethanol is the psychoactive substance identified most frequently in post-mortem specimens. Unfortunately, interpreting post-mortem ethanol concentrations can be difficult because of post-mortem alcohol redistribution and the possibility of postmortem alcohol neogenesis. Indeed, in the time interval between death and sample collection, the decedent may be exposed to non-controlled environments for an extended period, promoting microbial colonization. Many authors report that in the presence of carbohydrates and other biomolecules, various species of bacteria, yeast, and fungi can synthesize ethanol and other volatile substances in vitro and in vivo. The aim of this study was to study the impact of several variables on microbial ethanol production as well as develop a mathematical model that could estimate the microbial-produced ethanol in correlation with the most significant consensual produced higher alcohol, 1-propanol. An experimental setup was developed using human blood samples and cadaveric fragments incubated under strictly anaerobic conditions to produce a novel substrate, "cadaveric putrefactive blood" mimicking post-mortem corpse conditions. The samples were analyzed daily for ethanol and 1-propanol using an HS-GC-FID validated method. The formation of ethanol was evaluated considering different parameters such as putrefactive stage, blood glucose concentration, storage temperature, and storage time. Statistical analysis was performed using the Mann-Whitney non-parametric test and simple linear regression. The results indicate that the early putrefactive stage, high blood glucose concentration, high temperature, and time of incubation increase microbial ethanol production. In addition, the developed mathematical equation confirms the feasibility of using 1-propanol as a marker of post-mortem ethanol production. [ABSTRACT FROM AUTHOR]

Computed tomography (CT)-guided percutaneous needle biopsy of the lung is a well-recognized and relatively safe diagnostic procedure for suspicious lung masses. Systemic air embolism (SAE) is a rare complication of transthoracic percutaneous lung biopsies. Herein, we present a case of an 81-year-old man who underwent CT-guided percutaneous needle biopsy of a suspicious nodule in the lower lobe of the right lung. Shortly after the procedure, the patient coughed up blood which prompted repeat CT imaging. He was found to have a massive cardiac air embolism. The patient became unresponsive and, despite resuscitation efforts, was pronounced dead. The pathophysiology, risk factors, clinical features, radiological evidence, and autopsy findings associated with SAE are discussed, which may, in light of the current literature, assist with the dilemma between assessing procedural complications and medical liability. Given the instances of SAE in the setting of long operative procedures despite careful technical execution, providing accurate and in-depth information, including procedure-related risks, even the rarest but potentially fatal ones, is recommended for informed consent to reduce medicolegal litigation issues. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented in response to the article "Toward standardization of carbohydrate-deficient transferrin (CDT) measurement: I. Analyte definition and proposal of a candidate reference method" that was published in the previous issue.

Background: Interest has grown into the use of multidetector computed tomography (CT) and magnetic resonance imaging as an adjunct or alternative to the invasive autopsy. We sought to investigate these possibilities in postmortem CT scan using an innovative virtual autopsy approach. Methods: Twenty-five postmortem cases were scanned with the Philips Brilliance CT-64 and then underwent traditional autopsy. The images were interpreted by two blinded forensic pathologists assisted by a radiologist with the INFOPSY® Digital Autopsy Software System which provides three-dimensional images in Digital Imaging and Communications in Medicine format. Diagnostic validity of virtual autopsy (accuracy rate, sensitivity, specificity, and predictive values) and concordance between the two forensic pathologists (kappa intraobserver coefficients) were determined. Results: The causes of death at traditional autopsies were hemorrhage due to traumatic injuries (n = 8), respiratory failure (5), asphyxia due to drowning (4), asphyxia due to hanging or strangulation (2), heart failure (2), nontraumatic hemorrhage (1), and severe burns (1). In two cases, the cause of death could not be ascertained. In 15/23 (65%) cases, the cause of death diagnosed after virtual autopsy matched the diagnosis reported after traditional autopsy. In 8/23 cases (35%), traditional autopsy was necessary to establish the cause of death. Digital data provided relevant information for inferring both cause and manner of death in nine traumatic cases. The validity of virtual autopsy as a diagnostic tool was higher for traumatic deaths than other causes of death (accuracy 84%, sensitivity 82%, and specificity 86%). The concordance between the two forensic pathologists was almost perfect (>0.80). Conclusions: Our experience supports the use of virtual autopsy in postmortem investigations as an alternative diagnostic practice and does suggest a potential role as a screening test among traumatic deaths. [ABSTRACT FROM AUTHOR]

Although recent Cannabis use is widely reported to be associated with drug-related traffic accidents, the evidence that Cannabis users show an increased risk of being involved in road crashes is still not unequivocally proved. The purpose of the present work is to provide an objective assessment of this hypothesis, by comparing the frequency of occurrence of positive urine analyses in drivers involved in traffic accidents (n = 1406) with that observed in a control population undergoing mandatory urine drug testing (n = 1953). Urine analyses for drugs of abuse were performed by screening immunometric techniques followed by confirmation with UHPLC-QQQ MS, adopting a cut-off concentration for THC-COOH of 15 ng/mL. A case was classified as “positive” when a driver admitted to hospital for road traffic injuries showed urine concentrations of THC-COOH higher than the cut-off. All samples showing positive results for any other controlled drug in urine or blood alcohol concentrations >0.5 mg/mL were excluded from the study. Subjects positive to THC-COOH, and negative to all the other tested substances were 116 in Group 1 (8.2%) and 16 in Group 2 (0.8%). Subjects resulting negative to any tested substances were 1290 in Group 1 and 1937 in Group 2. The frequency of THC-COOH detection in the two groups was compared by using the “chi square” test, which resulted = 119.57, i.e. highly significant (P <<< 0.01). The Odds Ratio of the two groups was =10.88, showing a high degree of association between the presence of THC-COOH in urine and the occurrence of traffic accidents (P < 0.0001). The presented data, proving a high degree of association between Cannabis use and the occurrence of traffic accidents with injuries of the driver, support the use of urine testing for Cannabis in the procedures for the issuing of the driving licence, particularly in the case of subjects formerly or presently using Cannabis. This finding looks even more relevant in the present times, because of the increasing success of the policies of legalization of Cannabis for medical and non-medical purposes. [ABSTRACT FROM AUTHOR]

In this study, we compare the histological localisation of phenols and β-glucan in six cereal cross sections. The concentrations of β-glucan and total phenols in whole grain flour ( WGF) were also investigated to provide their nutrient content in grams. Cross sections of durum, soft, einkorn and emmer wheat, oats and barley were stained with Azan Trichrome ( ATH) and periodic acid-Schiff ( PAS) to characterise the morphology and cytohistology of the living cells in the aleurone, subaleurone and germ, as well as the a-nucleated cells in the starchy endosperm. Phenol localisation in the outer layers was performed by fluorescence microscopy due to the presence of ferulic acid. β-Glucan, examined with Calcofluor White staining, was found to be located primarily in the subaleurone layer and starchy endosperm of oats and barley. Our findings shed light on why WGF nutritional value is markedly reduced when the bran is removed through milling. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Ketamine, traditionally available as racemic mixture, has recently become available in the form of the single S-enantiomer, due to its higher anaesthetic potency associated with faster recovery times. The different pharmaceutical forms and the different pharmacodynamics of the two enantiomers imply the need for a chiral method, since most available analytical methods for biological matrices are not enantioselective. The method herein showed consists of simple capillary zone electrophoresis (CZE) for the chiral separation of ketamine and its major metabolite, norketamine, in hair specimens. After liquid-liquid extraction, the samples were electrokinetically injected and analysed in CE (running buffer: 15mM Tris phosphate pH 2.5, containing HS-γ-CDs, 0.1%, w/v). A complete separation of both racemic ketamine and norketamine in the respective enantiomers was obtained in less than 10minutes. Limit of detection (LOD) and limit of quantification (LOQ) were 0.08ng/mg and 0.25ng/mg, respectively. Percent recovery varied from 49% to 91% for all four enantiomers. Matrix effect on spiked hair samples demonstrated values ranging from 63% to 119%. Linearity was estimated using a calibration curve consisting of five concentration levels for each enantiomer (0.5-8.0ng/mg); the regression coefficients (R(2)) of weighted (1/x(2)) linear regression were all >0.988. The method is suitable for the analysis of real-world hair samples in order to investigate ketamine chronic abuse and to discriminate between the type of abused drug, either single enantiomer or racemic drug. [ABSTRACT FROM AUTHOR]

The natural mummy of Cangrande della Scala was exhumed from its tomb in the church of Santa Maria Antiqua in Verona and was submitted to a multidisciplinary study, that included archaeological, palaeopathological, palynological, toxicological and historical investigation. The body of Cangrande, still wearing his precious clothes, was in a good state of preservation. Palynological analyses demonstrated the presence of pollen grains of Digitalis sp./foxglove in the rectum content, along with Matricaria chamomilla /chamomille and Morus nigra /black mulberry. Toxicological analyses showed toxic concentrations of digoxin and digitoxin, two Digitalis glycosides, in the liver and faeces samples. Both palynological and toxicological data suggest an intoxication through the oral administration of an infusion or decoction of leaves and flowers of Digitalis . Cangrande died on July 22 1329, four day after his triumphal entrance in the city of Treviso. The sudden death was preceded by vomit and diarrhoea with fever that, according to written documents, he had contracted a few days before by “drinking from a polluted spring”. The gastrointestinal symptoms manifested by Cangrande in his last hours of life are compatible with the early phase of Digitalis intoxication and the hypothesis of poisoning is mentioned by some local historical sources. The palaeopathological analyses confirm a Digitalis poisoning. The most likely hypothesis on the causes of death is that of a deliberate administration of a lethal amount of Digitalis . Although several cases of poisoning through the use of organic substances are known from historical sources, no other direct evidences are documented in the palaeopathological literature. [ABSTRACT FROM AUTHOR]

Lithium salts are still one of the most popular therapeutic approaches to the treatment of bipolar disorders, notwithstanding the introduction of more modern, less toxic drugs. Because of a narrow therapeutic range, lithium serum concentrations must be strictly monitored during the treatment to avoid life-threatening neurotoxicity. For this purpose, methods based on flame photometry or ion-selective electrodes are usually applied. The aim of the present work was to develop and validate a simple method for the determination of lithium in serum based on capillary zone electrophoresis with indirect detection. A validation of the method was carried out, including a comparison with an automated routine method based on ion-selective electrodes. [ABSTRACT FROM AUTHOR]

Abstract: Identification of chronic excessive alcohol consumption in living and deceased individuals is a fundamental task in forensic pathology. Reliable methods for post-mortem diagnosis of chronic alcohol abuse are required because morphological findings are unspecific and ante-mortem data are often unreliable. In clinical practice, several biochemical markers indirectly demonstrating chronic alcohol abuse are employed, but thus far these methods have not been used in routine post-mortem investigations. We reviewed publications in which these markers have been applied to autopsy material. Based on this review, some of these biochemical parameters are useful in post-mortem diagnostics, although further systematic research is required. [Copyright &y& Elsevier]

Abstract: The coupling of capillary electrophoresis–electrospray ionization and time-of-flight mass spectrometry, combining efficiency and speed of separation with high mass accuracy and fast scanning capability, was for the first time applied to the determination of drugs of abuse (amphetamine, methamphetamine, MDA, MDMA, ephedrine, cocaine, morphine, codeine) and their metabolites in hair (6-MAM, benzoylecgonine). Experimental conditions were as follows. Separation: voltage 15kV, uncoated fused-silica capillary (75μm ID, 100cm total length), running electrolyte 25mM ammonium formate, pH 9.5, field-amplified sample stacking injection. Forensic drugs could be identified by exact mass determination (mass accuracy typically ≤ 5ppm) and by match of the isotopic pattern. The method was fully validated, showing limit of detections (LODs) suitable for the determination of all the compounds below the cut-off usually adopted for hair analysis (0.1ng/mg). Analytical precision in real matrices (tested at 0.1 and 1.0ng/mg) was typically characterized by CV''s ≤ 24% in both intra-day and day-to-day experiments. Quantitative determination was also tested by using a single internal standard (folcodine). Results, although with a moderate accuracy, conceivably depending on the lack of deuterated internal standards, proved useful for diagnostic use of the results from hair analysis. A single liquid–liquid extraction procedure was applied for all analytes, allowing the detection of a broad spectrum of basic drugs and their major metabolites. [Copyright &y& Elsevier]

Abstract: The purpose of the present work was the development and validation of a simple, rapid and reliable method for direct bromide quantification in serum based on capillary electrophoresis (CE). The analysis was carried out with an automated capillary electropherograph. Analytical conditions were as follows. Capillary: uncoated fused silica, effective length 50cm, internal diameter 50μm; voltage: 20kV in reverse polarity mode; temperature: 25°C; running buffer: 90mmol/L sodium tetraborate decahydrate and 10mmol/L NaCl, pH 9.24; detection: direct UV absorption at 200nm; sample treatment: dilution of serum 1:10 with the internal standard solution (2mmol/L thiocyanate). Under the described conditions, bromide ions and internal standard were baseline separated in 7min. No interferences from other serum components were observed. The analytical sensitivity was characterized by a LOD: 0.05mmol/L and a LOQ of 0.1mmol/L. Excellent linearity was verified in the range from 2.5 to 60mmol/L [y =0.0746x −0.0372; R 2 =0.9995 (x =bromide concentration; y =bromide peak area/internal standard (I.S.) peak area)]. Quantitative imprecision in intra-day (n =7) and day-to-day (n =7) experiments was always within R.S.D. values <2%. Recovery was quantitative throughout the range of linearity of the method. Clinical cases of infants undergoing potassium bromide therapy for refractory epilepsy were analyzed with results in agreement with literature data. On the basis of these considerations, capillary electrophoresis can be proposed as the method of choice for bromide analysis in serum samples, especially for therapeutic drug monitoring purposes. [Copyright &y& Elsevier]

Serum transferrin (Tf) comprises several isoforms with up to two complex oligosaccharide chains containing zero to eight sialic acid residues and neutral sugars. The major glycoform, known as tetrasialo-Tf, contains four sialic acid residues and accounts for about 80% of whole Tf in human serum. Carbohydrate-deficient transferrin (CDT) encompasses isoforms that are deficient in carbohydrate chains and consequently in sialic acid residues (including asialo-, monosialo- and disialo-Tf) and is a well known marker for chronic alcohol abuse. Recently capillary zone electrophoresis (CZE) has been reported as a tool extremely effective for the simultaneous, individual, quantitative determination of CDT isoforms. Three CZE methods that feature different dynamic capillary coatings were evaluated and optimized for CDT determination in human serum of alcohol abusers and control subjects. CZE separation was performed in alkaline borate buffers after serum sample saturation with iron, electropherograms were detected at 200 nm, data were evaluated as % area of disialo-Tf in relation to tetrasialo-Tf and peak identification was accomplished via relative migration times to tetrasialo-Tf, immunosubtraction and enzymatic sequential cleavage of sialic acid residues. Dynamic capillary coatings with diaminobutane, spermine and a double coating produced by commercially available proprietary agents were investigated and found to be suitable for determination of CDT in human serum. For all three approaches, best results were obtained in 50 μm I.D. fused-silica capillaries of 50 cm effective length and a capillary cartridge temperature of 20–25 °C. Using 3 mM 1,4-diaminobutane or 0.02 mM spermine in a borate-based running buffer of pH 8.3 provided data of remarkable similarity with resolution of di-, tri-, tetra- and pentasialo-Tf within 15–18 min. With the double coating, asialo-Tf and Tf isoforms with two to six sialic acid residues were baseline separated. Compared to the two amine-based procedures, the run times were found to be somewhat shorter, the detector signals higher, the applied power level significantly lower and the reproducibility better. [Copyright &y& Elsevier]

• CE-UV has been applied to the quantification of nitrite and nitrate in post-mortem specimen. • The method was applied to the quantitative determination of these ions in authentic forensic cases. • This is the first application of CE to the determination of nitrite/nitrate in fatalities. 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

• A Toxtyper™ LC-MSn screening method for the determination of drug in serum has been optimized. • The instrument performances have been verified on a panel of 47 drugs. • The method was applied to samples collected in the DUID context. 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. [ABSTRACT FROM AUTHOR]

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. Image 1 • A simple and low cost analytical method suitable for use at the crime scene for inferring the time since death is presented. • The device uses the microfluidic paper-based technology and the selectivity of the Nessler's reagent towards ammonium ion. • A concordance between the results obtained with the proposed procedure and those using CE-UV is proved. • The proposed procedure showed significant advantages for thanatochemical analysis at the crime scene. [ABSTRACT FROM AUTHOR]

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-days <15% and 20%, respectively. The results obtained analyzing 40 serum samples using the developed method, were compared with those obtained with HPLC-Vis and an R2 = 0.8854 was found. Considering 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. • The first analytical method for the CDT determination not requiring complex separative techniques nor immunoassays is described. • The proposed procedure is based on Fluorescence Resonance Energy Transfer (FRET) phenomenon. • A significant concordance between the results obtained with the proposed procedure and those using HPLC-Vis was proved. • The proposed procedure is potentially suitable for use as a Point-of-Care device. [ABSTRACT FROM AUTHOR]

Background: Although the post-mortem increase of ammonium in biological fluids is well known, ammonium analysis in vitreous humour has never been used in recent times for the determination of the post-mortem interval. The present work represents a new application of capillary electrophoresis with indirect UV detection in the field of forensic analysis. Methods: The electrophoretic separation was carried out in a running buffer made of 5 mM imidazole, 5 mM 18-crown-6 ether and 6 mM d,l-α-hydroxybutyric acid (HIBA). To overcome the lack of optical absorption of ammonium, indirect UV detection was applied. The used wavelength was 214 nm. Results: The method showed good linearity in the concentration range from 0.16 to 5.0 mM. The limit of detection, 0.039 mmol/L, was established on the basis of the linearity curve. Precision and bias studies carried out on the pure ammonium solutions and in real biological samples, revealed %RSDs well below 20%. A preliminary application to real cases where the death time was precisely known (14 bodies) was carried out plotting vitreous humour ammonium vs. post-mortem interval with a resulting good linear correlation until 100 h post-mortem. Conclusions: After validation in real cases, the present method can become a powerful tool to unravel one of the most challenging issues of forensic investigation: determination of the time of death. [ABSTRACT FROM AUTHOR]

Abstract Background and aim CDT is a collective biomarker including asialo- and disialo-Tf, but researchers have generally focused attention on disialo-Tf, because of its easier detectability, since asialo-Tf is typically not detectable by the current methods in abstinent individuals, social drinkers and in many alcohol abusers with moderate CDT increases. In the search of a confirmation marker of alcohol-related CDT increases, the detectability of asialo-Tf was re-evaluated comparatively by using CE vs. HPLC. Methods 468 serum samples compulsorily drawn in a forensic/administrative context were analyzed by CE and HPLC to compare their sensitivity towards asialo-Tf. Results CE allowed the identification of asialo-Tf in 108 out of 165 CDT "positive" cases, based on disialo-Tf measurement (cut-off 1.8%). HPLC showed a detectable asialo-Tf peak only in 2 cases. In addition, in some cases of disputed CDT increases, the quasi-absence of this Tf component in front of an important increase of disialo-Tf allowed the ruling out of a diagnosis of alcohol abuse, in agreement with all other clinical and laboratory data. Conclusions The present work shows a superior performance of CE vs. HPLC for the determination of asialo-Tf and the importance of this CDT component to avoid misinterpretation of non-alcohol related CDT increases. Highlights • CE and HPLC were compared in their ability to detect asialo-Tf. • CE showed a substantial prevalence over HPLC in the detection of asialo-Tf. • The results showed cases of discordant increases of disialo-Tf and asialo-Tf • In some of these cases the increase of CDT was not related with alcohol abuse [ABSTRACT FROM AUTHOR]

Background and aim The non-glycosylated glycoform of transferrin (Tf), known as asialo-Tf, was not selected (in favor of disialo-Tf) as the measurand for the standardization of carbohydrate deficient transferrin (CDT) determination because of a lower diagnostic sensitivity provided with the currently available analytical procedures for sera. However, asialo-Tf could provide an additional value to disialo-Tf in the CDT analysis employed in forensic toxicology contexts. The present work aimed at developing an easy sample preparation based on PEG precipitation in order to improve the detectability of asialo-Tf in capillary electrophoresis (CE). Methods Equal volumes (35 μL) of serum and of 30% PEG-8000 were mixed and briefly vortexed. After centrifugation, the supernatant was iron saturated with a ferric solution (1:1, v/v). The mixture was analyzed in CE for asialo-Tf and disialo-Tf determination. Results PEG-8000 precipitation allowed the improvement of the baseline in the electropherograms in terms of interferences reduction particularly in the asialo-Tf migration region. The detection of asialo-Tf was possible in 89% of samples with disialo-Tf above the cut-off limit, whereas only 16% of them showed asialo-Tf by employing the traditional sample preteatment. Conclusions Asialo-Tf represents an additional value to disialo-Tf as a biomarker of alcohol abuse in forensic toxicology. [ABSTRACT FROM AUTHOR]

The aim of the present work was to develop a novel in-house mixed-mode SPE sorbent to be used for the HPLC-Ion TrapMS determination of 16 basic drugs in urine. By using a computational modelling, a virtual monomer library was screened identifying three suitable functional monomers, methacrylic acid (MAA), itaconic acid (IA) and 2-acrylamide-2-methylpropane sulfonic acid (AMPSA), respectively. Three different sorbents were then synthetized based on these monomers, and using as cross-linker trimethylolpropane trimethacrylate (TMPTMA). The sorbent characterization analyses brought to the selection of the AMPSA based phase. Using this novel in-house sorbent, a SPE-HPLC-Ion TrapMS method for drug analysis in urine was validated proving to be selective and accurate and showing a sensitivity adequate for toxicological urine analysis. The comparison of the in-house mixed-mode SPE sorbent with two analogous commercial mixed-mode SPE phases showed that the first one was better not only in terms of process efficiency, but also in terms of quality-price rate. To the best of our knowledge, this is the first time in which an in-house SPE procedure has been applied to the toxicological analysis of a complex matrix, such as urine. [ABSTRACT FROM AUTHOR]

This paper puts forward a new method for the transferrin (Tf) glycoform analysis in body fluids that involves the formation of a transferrin-terbium fluorescent adduct (TfFluo). The key idea is to validate the analytical procedure for carbohydrate-deficient transferrin (CDT), a traditional biochemical serum marker to identify chronic alcohol abuse. Terbium added to a human body-fluid sample produced TfFluo. Anion exchange HPLC technique, with fluorescence detection ( λ 298 nm and λ 550 nm), permitted clear separation and identification of Tf glycoform peaks without any interfering signals, allowing selective Tf sialoforms analysis in human serum and body fluids (cadaveric blood, cerebrospinal fluid, and dried blood spots) hampered for routine test. Serum samples ( n = 78) were analyzed by both traditional absorbance (Abs) and fluorescence (Fl) HPLC methods and CDT% levels demonstrated a significant correlation ( p < 0.001 Pearson). Intra- and inter-runs CV% was 3.1 and 4.6%, respectively. The cut-off of 1.9 CDT%, related to the HPLC Abs proposed as the reference method, by interpolation in the correlation curve with the present method demonstrated a 1.3 CDT% cut-off. Method comparison by Passing-Bablok and Bland-Altman tests demonstrated Fl versus Abs agreement. In conclusion, the novel method is a reliable test for CDT% analysis and provides a substantial analytical improvement offering important advantages in terms of types of body fluid analysis. Its sensitivity and absence of interferences extend clinical applications being reliable for CDT assay on body fluids usually not suitable for routine test. [ABSTRACT FROM AUTHOR]

The relevance of the problem of urine tampering is well-known in forensic toxicology, with sample dilution being the most used method to cheat toxicological controls. Among the criteria to assess urine integrity, the quantification of creatinine probably represents the most popular method. The present paper presents a simple and low-cost analytical device for on-site creatinine determination as first-line screening for urine dilution. The proposed microfluidic devices were designed as a three-dimensional origami pattern. The device included three colorimetric reactions based on picric acid (PA-based reagent), 3,5-dinitrobenzoic acid (DNBA-based reagent), and Nessler's reagent. The last one, to the best of our knowledge, has never been used before for creatinine determination. In order to assure the highest ease and economy of operation, the color detection and data processing were performed using a built-in smartphone camera and the associated software. The optimized device showed a detection limit of 0.02 g/L. The proposed method was used for the qualitative screening for urine dilution of 48 samples, showing a diagnostic sensitivity and specificity for PA-based, DNBA-based and Nessler's reagent of 83.3%–80.0%, 72.2%–70.0%, and 100.0%–93.3% respectively, versus reference enzymatic method adopting a cut-off of 0.2 g/L. In conclusion, the present preliminary study shows that the proposed device could be a useful tool for on-site screening for urine tampering at the time of sample collection for toxicological testing. [Display omitted] • A rapid and low-cost analytical method suitable for the on-site assessment of urine tampering is presented. • The proposed device is based on an origami paper-based microfluidics technology (3D-μPAD). • The 3D-μPAD integrates three reagents, including Nessler's reagent, for the semiquantitative determination of creatinine. • The proposed procedure showed significant advantages for the verification of urine integrity. • Nessler's reagent offers higher performances than picric acid-based and dinitrobenzoic acid-based reagents. [ABSTRACT FROM AUTHOR]

Background: 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.Methods: 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 (BAC>0.50 g/L); all drivers were also tested for CDT.Results: 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.Conclusions: 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". [ABSTRACT FROM AUTHOR]

Cases of subtle fatal neck compression are often complicated by the lack of specificity of the post-mortem signs of asphyxia and by the lack of clear signs of neck compression. Herein we present a forensic case of a 45-year-old schizophrenic patient found on the floor of the bedroom of a psychiatric ward in cardiopulmonary arrest and who died after two days in a vegetative state. The deposition of the roommate of the deceased, who claimed responsibility for the killing of the victim by neck compression, was considered unreliable by the prosecutor. Autopsy, toxicological analyses, and multi-slice computed tomography (MSCT), micro computed tomography (micro-CT) and histology of the larynx complex were performed. Particularly, micro-CT analysis of the thyroid cartilage revealed the bilateral presence of ossified triticeous cartilages and the complete fragmentation of the right superior horn of the thyroid, but it additionally demonstrated a fracture on the contralateral superior horns, which was not clearly diagnosable at MSCT. On the basis of the evidence of intracartilaginous laryngeal hemorrhages and bilateral microfracture at the base of the superior horns of the larynx, the death was classified as a case of asphyxia due to manual strangulation. Micro-CT was confirmed as a useful tool in cases of subtle fatal neck compression, for the detection of minute laryngeal cartilage fractures, especially in complex cases with equivocal findings on MSCT. [ABSTRACT FROM AUTHOR]

Abstract: Capillary electrophoresis coupled to time-of-flight mass spectrometry was used in the present work for the determination of therapeutic and abused drugs and their metabolites in the hair of subjects undergoing addiction treatments, in order to monitor their compliance to therapy. For this purpose a rapid, qualitative drug screening method was adopted based on capillary electrophoresis hyphenated with time-of-flight mass spectrometry, which had earlier been developed and validated for the forensic-toxicological analysis of hair, limitedly to illicit/abused drugs . Sampling of hair was carried out in order to refer to a time window of about two months from the date of sampling (i.e. 2cm ca. from cortex). A single extraction procedure was applied, allowing the determination in the hair matrix of “drugs of abuse” referred to the past abuses, and therapeutic drugs prescribed in the detoxification program as well as their metabolites. Analyte identification was based on accurate mass measurements and comparison of isotope patterns, providing the most likely matching between accurate mass value and elemental formula. Small molecules (<500Da) of forensic and toxicological interest could be identified unambiguously using mass spectrometric conditions tailored to meet a mass accuracy ≤5ppm. In the present study, the proposed approach proved suitable for the rapid broad spectrum screening of hair samples, although needing further confirmation of results by using fragmentation mass spectrometry. [Copyright &y& Elsevier]

The article focuses on the multielemental analysis of Verona Prince Cangrande della Scala's tissue specimens to determine the real cause of his death. It notes that inductively coupled plasma-mass spectrometry (ICP-MS) analysis was performed to hair and tissue samples. Results contradict the notion that the Prince's death was due to arsenic poisoning since the element showed very low concentrations in the analyzed samples. It also notes that the Prince was exposed to lead during his lifetime.

Abstract: In this study, we present a capillary electrochromatographic method for separation of basic compounds of interest in forensic science (amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxyethylamphetamine, cocaine, codeine, heroin, morphine, and 6-monoacethylmorphine). Several analytical conditions were taken into account to completely separate in the same run the 10 drugs of abuse analyzed. Chromatographic retention, selectivity and efficiency were evaluated in dependence of the type of stationary phase (CN and RP-C18 derivatized silica particles), mobile phase composition, buffer type and pH, sample injection. The optimum separation parameters were set up using a mixture of aqueous sodium phosphate buffer (pH 2.5)/acetonitrile (80/20, v/v) as the mobile phase, 10kV and 20°C as applied voltage and capillary temperature, respectively. Under these conditions all the studied analytes were baseline resolved within 20min. The method performance was investigated in terms of precision, linearity, sensitivity and accuracy to demonstrate the applicability of the developed capillary electrochromatographic system to forensic analysis. Calibration curves provided a good linearity over a working range of 100–1200ng/mL for all analytes. Limits of detection and quantification were in the range 5–12ng/mL and 10–30ng/mL, respectively. Then the method was applied to the analysis of a human urine sample spiked with a basic compounds’ mixture. Urine samples’ pre-treatment was carried out through a solid phase extraction (SPE) procedure on strong cation exchange (SCX) cartridges. [Copyright &y& Elsevier]

Abstract: After decades of neglect, bromide has recently been re-introduced in therapy as an effective anti-epileptic drug. The present paper describes the methodological optimization and validation of a method based on capillary zone electrophoresis for the rapid determination of bromide in serum using a high-viscosity buffer and a short capillary (10cm). The optimized running buffer was composed of 90mM sodium tetraborate, 10mM sodium chloride, pH 9.24 and 25% glycerol. The separation was carried out at 25kV at a temperature of 20°C. Detection was by direct UV absorption at 200nm wavelength. The limit of detection (signal-to-noise ratio=5) in serum was 0.017mM. The precision of the method was verified in blank serum samples spiked with bromide, obtaining intra-day and day-to-day tests, relative standard deviation values ≤0.2% in terms of migration times and values <2% in terms of peaks areas, respectively. [Copyright &y& Elsevier]

Abstract: Post-mortem diagnosis of chronic alcohol abuse is a challenge for forensic experts due to the lack of pathognomonic morphological findings and often also inadequate background information. Objective methods demonstrating chronic excessive alcohol consumption would therefore be a useful tool for forensic pathologists. In clinical practice, several markers of chronic alcohol abuse have recently been introduced, among which carbohydrate-deficient transferrin (CDT) is the most accepted, but the use of these markers in autopsy has not yet been established. We examined post-mortem stability and possible post-mortem redistribution of CDT and compared two analytical methods, capillary zone electrophoresis and high-performance liquid chromatography. According to our results, CDT remains stable for an appreciable time after death. The results further indicate that CDT is not subject to major post-mortem redistribution. [Copyright &y& Elsevier]

Abstract: Alpha-bisabolol is a compound present in some essential oils, widely distributed in several plants, including camomile. Two different methods for analysing an essential oil, such as alpha-bisabolol in human blood are reported: the first uses micro-liquid chromatography–electrospray ionisation-mass spectrometry (μHPLC–ESI-MS), whereas the second is based on “head space” injection coupled to gas chromatography–mass spectrometry (HS-GC–MS). For LC–ESI-MS, human blood samples, spiked with alpha-bisabolol, were extracted with hexane and evaporated to dryness under air stream. The residue was then reconstituted with methanol and injected into a C18 column, connected to an ion trap mass spectrometer equipped with an ESI source. Spectra were recorded in the positive ion, selected ion monitoring mode. The detection limit of alpha-bisabolol in blood was 0.125μmol/l. The preparation of samples for the analysis in HS-GC–MS was limited to blood dilution with water (0.5ml blood + 1ml water). Head space vials were heated at 125°C for 1h before automatic injection. The HS-GC–MS detection limit (0.13μmol/l) was similar to that achieved with the μHPLC–ESI-MS method. Successful tests were performed to verify if alpha-bisabolol could be directly measured by the HS-GC–MS method in different biological samples (blood, urine, faeces, homogenate tissues) from rats treated with the camomile essential oil. [Copyright &y& Elsevier]

Carbohydrate deficient transferrin (CDT) is currently the most specific laboratory marker of chronic or sustained alcohol abuse. CDT is increasingly being used as a diagnostic tool in the areas employment, traffic safety and forensic medicine. In recent times, capillary electrophoresis (CE) has been proposed as a convenient tool for rapid, precise and accurate CDT determination, not only for research but also for routine analyses. Quite recently, commercial kits have been introduced which, reportedly, could simplify and standardize CDT analysis with capillary electrophoresis. The present work was aimed at testing the ruggedness of a capillary electrophoretic method based on a commercial kit (CEofix, Analis), by comparing the results obtained with different instruments in different laboratories, on a panel of sera randomly collected and exchanged. The results showed, notwithstanding few outliers, excellent correlation of the results obtained in the two laboratories (R=0.974). Also high concordance was found when results were classified as positive or negative on the basis of a cut-off (1.25%) established from a control group of teetotalers. In conclusion the present data support the usefulness of capillary electrophoresis for CDT determination for clinical, forensic and administrative diagnosis of chronic alcohol abuse. [Copyright &y& Elsevier]

The chemical and biochemical analysis of bodily fluids after death is an important thanatochemical approach to assess the cause and time since death. Vitreous humor (VH) has been used as a biofluid for forensic purposes since the 1960s. Due to its established relevance in toxicology, a literature review highlighting the use of VH with an emphasis on endogenous compounds has not yet been undertaken. VH is a chemically complex aqueous solution of carbohydrates, proteins, electrolytes and other small molecules present in living organisms; this biofluid is useful tool for its isolated environment, preserved from bacterial contamination, decomposition, autolysis, and metabolic reactions. The post-mortem analysis of VH provides an important tool for the estimation of the post-mortem interval (PMI), which can be helpful in determining the cause of death. Consequently, the present review evaluates the recent chemical and biochemical advances with particular importance on the endogenous compounds present at the time of death and their modification over time, which are valuable for the PMI prediction and to identify the cause of death. [ABSTRACT FROM AUTHOR]