European Council of Legal Medicine (ECLM) on-site inspection forms for forensic pathology, anthropology, odontology, genetics, entomology and toxicology for forensic and medico-legal scene and corpse investigation: the Parma form.

Further to a previous publication by the European Council of Legal Medicine (ECLM) concerning on-site forensic and medico-legal scene and corpse investigation, this publication provides guidance for forensic medical specialists, pathologists and, where present, coroners' activity at a scene of death inspection and to harmonize the procedures for a correct search, detection, collection, sampling and storage of all elements which may be useful as evidence, and ensure documentation of all these steps. This ECLM's inspection form provides a checklist to be used on-site for the investigation of a corpse present at a crime or suspicious death scene. It permits the collection of all relevant data not only for the pathologist, but also for forensic anthropologists, odontologists, geneticists, entomologists and toxicologists, thus supporting a collaborative work approach. Detailed instructions for the completion of forms are provided.

Driving under the influence of drugs: Prevalence in road traffic accidents in Italy and considerations on per se limits legislation.

OBJECTIVE: The objective of this study was to present the prevalence and concentrations of drugs in blood samples of drivers involved in road traffic accidents (RTAs) and to discuss the effects of adopting different concentration cutoff values proposed or applied in other European countries on the number of driving under the influence of drugs (DUID) offenses. METHODS: Blood samples from drivers involved in RTAs in Padova province from 2014 to 2017 were analyzed for the presence of alcohol and drugs. The prevalence of positive subjects was reported for each substance adopting the limits of quantification (LOQs) of our laboratory and the concentration cutoff values proposed and/or used in other European countries. The reduction of cases of driving under the influence of illicit drugs in applying different cutoffs was calculated. RESULTS: Four thousand four hundred forty-three blood samples were analyzed: 23.7% were positive for alcohol and 19.9% for psychoactive drugs, with prevalences of polydrug and alcohol-drug abuse of 4.5 and 6%, respectively. The most frequently detected drugs were cannabinoids (9.7%) and cocaine (7.2%), followed by benzodiazepines (4.1%), opiates (1.9%), and other opioids (1.7%). Barbiturates, amphetamines, and ketamine were identified in a much smaller number of cases. The overall decrease in DUID cases when adopting different cutoffs with respect to cases above the LOQs was between 8 and 84%. The adoption of high LOQs such as those used in the European Union's research project on Driving Under the Influence of Drugs, Alcohol and Medicines (DRUID) decreases the hypothetical number of DUID offenses by a quarter, and per se limits proposed as broadly equivalent to a blood alcohol concentration (BAC) between 0.2 and 0.8 g/L dramatically reduce the cases of DUID (cocaine -81%, cannabis -79%, opioids -97%, opiates -96%, and amphetamines -77%); no ketamine-positive samples were above the cutoff. CONCLUSIONS: The implementation of high analytical limits or per se limits based on impairing concentrations in the Italian legislation could result in the prosecution of a much lower number of drugged drivers involved in RTAs, with a decrease from 25% to more than 80% depending on the limits.

Occupational exposure to ketamine detected by hair analysis: a retrospective and prospective toxicological study.

Ketamine (KT) is used to induce and maintain general anaesthesia in combination with sedative drugs in human and animals. Because of its dissociative and hallucinogenic effects, KT has become a recreational drug in a variety of social settings and may be included in the panel of drugs of abuse that are controlled in driving under the influence (DUI) ascertainment. In a local driving license re-granting protocol, a case where a veterinary physician was found positive to KT and nor-ketamine (NK) in hair suggested the possibility of a professional exposure in a veterinary setting and prompted an experimental study. Male (7) and female (4) veterinary physicians were recruited on a voluntary base. Detailed information was collected on their habits, use of drugs, professional practice, frequency and mode of using KT injections. Hands and skin were examined. Head hair and pubic hair were collected. Two naïve subjects, starting their professional practice at a local veterinary clinic, were recruited and their hair (head, pubic, axillary, thoracic hair, and beard) and urine were collected before and after usual clinic activity. Hair were cut according to their length, washed, pulverized and 25mg were extracted and analyzed by liquid chromatography coupled to high accuracy, high resolution mass spectrometry. All the hair samples from the veterinary physicians turned to be positive for KT, at a concentration varying from 0.010 to 0.840ng/mg in head hair and from 0.040 to 2.04ng/mg in pubic hair; NK ranged from not detected to 0.080ng/mg in head hair, from not detected to 0.100 in pubic hair; when KT was ≥0.100, NK was always detected. For the two naïve subjects, hair from different body sites were negative before they started their activity, and positive one month later; some urine samples resulted positive and confirmed systemic exposure to KT. The possibility of unaware exposure to KT was demonstrated. The site of absorption is skin, independently from the presence of skin injuries due to hand contact with KT injection solutions and/or animal body fluids by veterinary physicians during clinical activities and animal handling. Possible adverse systemic effects by unaware KT exposure need to be studied and concerns have arisen as to healthy and safe workplace policies. Comparison with a population of subjects undergoing driving license re-granting evidenced a larger range of concentration in this latter setting (0.050-10.0ng/mg for KT, not detected to 0.100ng/mg for NK).

In vitro sensitivity to methyl-prednisolone is associated with clinical response in pediatric idiopathic nephrotic syndrome.

The aim of this study was to evaluate the in vitro steroid sensitivity as a predictor of clinical response to glucocorticoids in childhood idiopathic nephrotic syndrome (INS). Seventy-four patients (median age 4.33, interquartile range [IQR] 2.82-7.23; 63.5% male) were enrolled in a prospective multicenter study: in vitro steroid inhibition of patients' peripheral blood mononuclear cell proliferation was evaluated by [methyl-(3) H] thymidine incorporation assay at disease onset (T0) and after 4 weeks (T4) of treatment. Steroid dependence was associated with increased in vitro sensitivity at T4 assessed both as drug concentration inducing 50% of inhibition (IC50 ; odds ratio [OR] = 0.48, 95% confidence interval [CI] = 0.24-0.85; P = 0.0094) and maximum inhibition at the highest drug concentration (Imax ; OR = 1.13, 95% CI = 1.02-1.31; P = 0.017). IC50 > 4.4 nM and Imax < 92% at T4 were good predictors for optimal clinical response. These results suggest that this test may be useful for predicting the response to glucocorticoid therapy in pediatric INS.

Matrix-assisted laser desorption/ionization mass spectrometry, enzymatic digestion, and molecular modeling in the study of nonenzymatic glycation of IgG.

The glycation-induced functional change of immunoglobulins is of particular interest. The glycation levels of IgG in 10 healthy subjects and 20 diabetic patients with different degrees of metabolic control were studied by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. It reveals the number of glucose molecules that have condensed on the protein, which range from 1 to 5 for healthy subjects, from 5 to 9 for well controlled diabetic patients, and from 10 to 25 for poorly controlled ones. The identification of the most favored glycation sites has been obtained by MALDI analysis of standard and in vitro glycated IgG and plasma protein fraction of a healthy subject after digestion with papain, releasing Fab and Fc fragments of the molecule. Both experiments, as well as molecular modeling of the whole protein, confirm that the most of glucose molecules have condensed on the Fab fragment of IgG, suggesting that the immune deficiency observed in diabetic patients may be explained at the molecular level by a more effective glycation of the Fab fragment, thus inhibiting the process of molecular recognition between antibody and antigen.

Application of matrix-assisted laser desorption/ionization mass spectrometry to the detection of melanins formed from Dopa and dopamine.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry was used to study melanogenesis starting from Dopa and dopamine, the latter considered one of the precursors of neuromelanins. These substrates were left to react with the peroxidase - H(2)O(2) system, which is postulated to play an important role in melanin biosynthesis. Samples were prepared by ultrafiltering the substrate - enzyme solution after 30, 60, 120, 240 and 360 min of reaction and aliquots were immediately lyophilized. The reaction of dopamine with peroxidase - H(2)O(2) favoured the formation of dopamine oligomers up to octamers. In contrast, the action of either peroxidase or H(2)O(2) alone, studied for comparison, did not lead to melanin production and only dimeric and trimeric species were observed. Also for Dopa, analogous results were obtained in the presence of either peroxidase or H(2)O(2) alone, without melanin formation. Conversely, Dopa with the peroxidase - H(2)O(2) system led to the formation of a black precipitate after 120 min of reaction, and oligomers of 5,6-dihydroxyindole (DHI), an intermediate of melanogenesis, were detected, together with products of further oxidation. Faster kinetics were observed when Dopa was treated with tyrosinase, the enzyme catalysing the oligomerization of tyrosine to melanins, leading to the formation mainly of DHI oligomers.

Advanced glycation end products: a highly complex set of biologically relevant compounds detected by mass spectrometry.

Structural information on 'AGE-peptides,' a class of substances belonging to advanced glycation end products (AGE) and originating by proteolysis of glycated proteins, was gained through various analytical approaches on the mixture produced by proteinase K digestion of in vitro glycated bovine serum albumin. Both matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS) were employed, and the results were compared with those from conventional spectroscopic methods (UV, fluorescence, gel permeation). The data acquired by the various techniques all depict the digestion mixtures as highly complex, with components exhibiting molecular mass in the range 300-3500 Da. In the analysis of HPLC/ESI-MS data, identification of AGE-peptides was facilitated by 3D mapping. Structural information was gained by means of multiple mass spectrometric experiments.

Evidence of gold-gold intra- and intermolecular interactions gained by laser desorption/ionisation and fast atom bombardment mass spectrometry in the study of some gold(I) coordination compounds

The mass spectrometric behaviour of some two-coordinated linear gold(I) complexes has been studied by fast atom bombardment (FAB) and laser desorption/ionisation (LDI) methods. The results obtained demonstrate some interesting aspects of Au-Au interactions. The detection under FAB conditions of Au(n)(+) clusters, in particular of Au(3)(+) species, can be rationalised by the presence of significant intramolecular Au-Au interactions, while the formation under LDI conditions of Au(n)(+) clusters can be considered indicative of Au(ellipsis)Au intermolecular interactions present in the solid state. Copyright 2000 John Wiley & Sons, Ltd.

Ion trap mass spectrometry, a new tool in the investigation of drugs of abuse in hair.

The use of an ion trap mass spectrometer for the detection, via tandem mass spectrometry, of morphine and cocaine in the hair of drug addicts proved to be successful. In particular, detection limits of 1-20 ppb for morphine and 5-30 ppb for cocaine were achieved, with good reproducibility and absence of interferent species.

Melanogenesis from 5-hydroxytryptamine, 5,6- and 5,7-dihydroxytryptamines. An in vitro study using MALDI-TOF.

The role of tyrosinase and peroxidase in melanogenesis of 5-hydroxytryptamine, 5,6- and 5,7-dihydroxytryptamines was investigated by matrix-assisted laser desorption/ionization mass spectrometry. Each enzyme was incubated with the tryptamine derivatives and samples were drawn at various times, ultrafiltered and immediately lyophilized. The results indicated that peroxidase promotes oligomerization of 5-HT with fast kinetics but with yields lower than those achieved by tyrosinase. 5,6- and 5,7-DHT formed low molecular mass oligomers in the presence of peroxidase alone. The addition of hydrogen peroxide evidences different reactivity of the two isomers: 5,6-DHT formed immediately a black precipitate while oligomers of the molecule itself and of its oxidation products were detectable for 5,7-DHT.

Unimolecular decomposition of M+. and [M+H]+ species of some fluorosubstituted acyclic nucleoside analogs.

Electron ionization and fast-atom bombardment mass spectrometry are shown to provide a valid analytical tool for the structural characterization of the title compounds. In fact, diagnostic fragmentation pathways were observed depending on the presence of different substituents (benzyloxy, (benzyloxy, p-tolylthio, p-tolylsulphinyl) as well as of different bases. Regioisomeric compounds could be differentiated by kinetic energy release measurements.

Silicon membrane interface for the direct analysis of Kathon CG in aqueous solutions and cosmetic emulsions.

An easy determination of Kathon CG, an antimicrobial agent widely used in cosmetic and toiletry products, has been achieved by means of silicone membrane interfaced mass spectrometry. Low levels of the above preservative (up to p.p.b.) could be easily detected in both aqueous solutions and cosmetic emulsions. Valuable information has been obtained on the stabilization and decomposition processes to which the organic components of Kathon CG are subject. The role of magnesium ions, which are present as a stabilizing agent in Kathon CG formulation, in these processes has been investigated, leading to the identification of some degradation products.

Monitoring the production yields of vincristine and vinblastine in Catharanthus roseus from somatic embryogenesis. Semiquantitative determination by flow-injection electrospray ionization mass spectrometry.

A semiquantitative determination of two bis-indole antitumor alkaloids, vincristine and vinblastine, has been performed by flow-injection electrospray ionization mass spectrometric analysis of the extracts of Catharanthus roseus. Leaves and flowers of two different phenotypes (pink flower and white flower) obtained from somatic embryogenesis were thus examined and compared with the field-grown mother plant. Different amounts of vincristine and vinblastine were detected depending on the examined samples.

Mass spectrometry in the study of non-enzymatic glyco-oxidation of proteins.

Studies that have been performed by MALDI mass spectrometry in the field of non-enzymatic protein glyco-oxidation are briefly reviewed. The results obtained by in vitro and in vivo experiments demonstrate the power of this technique, which is able to give information on the number of sugar molecules condensed on specific proteins and, consequently, represents a new and specific diagnostic tool in diabetes control.

Effects of ultraviolet irradiation on melanogenesis from tyrosine, Dopa and dopamine: a matrix-assisted laser desorption/ionization mass spectrometric study.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry experiments were applied to study the influence of ultraviolet (UV) irradiation in melanogenesis. Samples were prepared starting from three different precursors, tyrosine, Dopa and dopamine, in the presence or absence of tyrosinase, the enzyme responsible for the synthesis of melanin. Enzymatic reactions were carried out for 10, 30, 60 and 120 min under UV irradiation at 365 nm, and aliquots were then immediately ultrafiltered and lyophilized. Samples obtained by irradiation of tyrosine solution revealed the formation of 5,6-dihydroxyindole (DHI) oligomers up to pentamers at 120 min; the reaction kinetics were markedly enhanced in the presence of tyrosinase. In the case of Dopa, UV irradiation favored melanogenesis only in the presence of the enzyme; in this case, many reaction pathways were activated, originating various oligomeric species of Dopa, DHI and 5,6-dihydroxyindole-2-carboxylic acid (DHICA). Conversely, when dopamine was used as tyrosinase substrate under UV light, mechanisms of melanogenesis different from those generated by simple enzymatic reaction without irradiation were not activated, as the same oligomeric species were present.

Characterization of hematology lysing reagents containing quaternary ammonium halides using fast-atom bombardment mass spectrometry.

Positive- and negative-ion fast-atom bombardment (FAB) mass spectrometry has been employed to characterize pure dodecyl-, tetradecyl- and hexadecyl-trimethylammonium chlorides and bromides, compounds of high interest as lysing agents in hematologic analyses. In parallel, the same approach has been used for some commercially available lysing reagent products. While in positive-ion FAB mass spectra the presence of the abundant ions corresponding to the intact quaternary ammonium cations (Q+) is accompanied by the presence of [Q2X]+ (X = Cl, Br) species, in the negative-ion mode cluster ions such as [QX2]- are the sole ionic species that are easily evident. The mechanism for the formation of [Q2X]+ and [QX2]- species has been studied and some suggested explanations are given.

Collisional spectroscopy approaches in the characterization of 7-alpha- and 7-beta-hydroxycholesterol.

High- and low-energy collision experiments, by means of double-focusing and ion-trap mass spectrometers, respectively, performed on the M+ and [M-H2O]+. ions of 7-alpha- and 7-beta-hydroxycholesterol lead to a clear structural characterization of the two stereoisomers. In particular, the beta isomer shows peculiar fragmentation pathways due to interaction between the hydroxyl group in position 7-beta and the hydrogen atom on C(15).

High-performance liquid chromatographic and mass spectrometric identification of phosphocitrate synthesized by human kidney homogenate.

Two high-performance liquid chromatographic techniques for the analysis of phosphocitrate, a metabolite synthesized by the cell that is thought to play a role in a number of physiological as well as pathological events, have been developed. The mass spectra of the isolated compound obtained under fast-atom bombardment and collisionally activated decomposition mass-analysed ion kinetic energy conditions are also reported.

Curie-point pyrolysis-gas chromatography/mass spectrometry in the art field. 2--The characterization of proteinaceous binders.

Curie point pyrolysis with gas chromatography/mass spectrometry has been employed to characterize some proteinaceous media used in the art field as painting binders: milk casein, egg yolk, egg albumin, bone glue, skin glue, rabbit glue and fish glue. A careful analysis of the gas chromatograms so obtained has led to the distinction of the different proteinaceous binders in terms of different chromatographic profiles. Some of the pyrolysis products have been identified by library search.