Chemical investigation of biological trace evidence; toxicological screening of waste residues obtained from DNA extraction processes.

In a forensic scenario, if biological stains are found in very small quantities, these are usually collected for DNA analyses, considered essential for the forensic investigation and thus excluding possible investigations by other forensic disciplines as forensic toxicology. We developed an experimental study to evaluate the feasibility of analyzing DNA extraction residues obtained from DNA extraction procedures to perform toxicological analysis, with the aim to extract both genetic and toxicological information without affecting or compromising the genetic sample and/or DNA extraction. DNA extraction from four blood samples (fortified with 5 molecules of interest with a final concentrations of 1 µg/mL, 100 ng/mL, 10 ng/mL and 5 ng/mL, respectively) were analyzed with QIAGEN QIAmp® DNA Mini kit. Three waste residues collected from the DNA extraction were analyzed for the toxicological investigation via Solid-Phase Extraction and High-Performance Liquid Chromatography-Tandem Mass Spectrometry analyses (Thermo Scientific™ TSQ Fortis™ II Triple-Quadrupole Mass Spectrometer). The analytical investigation revealed that our analytes of interest were detected in two different residues of the DNA extraction procedure, allowing both genetic and toxicological analyses without affecting the DNA identification. At last, the experimental protocol was applied to a hypothetical case, with encouraging results and allowing the identification of our molecules of interest.

Water-related atmospheric agents and solubility: two parameters of validation in toxicological screening on clothing worn by skeletal remains.

In forensic toxicology, when conventional matrices are no longer available, alternative matrices can be used to assess toxicological investigations. Clothes worn by skeletal remains may be a good unconventional matrix for toxicological analyses considering that they have absorbed decomposition fluids and blood from a body. We hypothesized a scenario in which a skeleton, wearing clothes, was discovered in an open environment. From this starting point, an experimental study was developed on different textiles (cotton, wool, and polyester) to evaluate whether water-related atmospheric agents and molecule solubility can largely influence the detection of molecules of toxicological interest on this specific matrix, together with the characteristics of different garments chosen. The experimental study was performed on blood spots, previously spiked with 6-monoacetylmorphine and morphine, accurately placed on different textiles and washed with different quantities of deionized water adjusted at pH 5.6 with formic acid to simulate different rainfall conditions. Toxicological analyses were performed via Solid-Phase Extraction and High-Performance Liquid Chromatography-Tandem Mass Spectrometry analyses (Thermo Scientific™ TSQ Fortis™ II Triple-Quadrupole Mass Spectrometer). From the experimental study morphine could not be detected on 100% cotton and 100% wool fabric after the passing of 500 mL of deionized water and in 100% synthetic polyester textile after washing with 250 mL of deionized water. In conclusion, when toxicological analyses are carried out on unconventional matrices as textiles worn by corpses exposed to different environmental conditions, it is of great importance, in using such substrates as evidence for the presence of molecules of toxicological interest, to evaluate chemical-physical characteristics of each analyte under investigation in order to correctly interpret the toxicological data obtained.

A unique case of death by MDPHP with no other co-ingestion: a forensic toxicology case.

Synthetic cathinones are a class of psychoactive drugs that have become, in recent years, of quite common observation in medical and toxicological forensic field. MDPHP (3,4-methylenedioxy-α-pyrrolidinohexanophenone) belongs to this class of substances but lethal acute intoxication caused by this specific substance without other co-ingestions has never been described in literature. We are presenting the unique case of a fatal acute intoxication caused by MDPHP in a 48-year-old man carried to the A&E department of a big Milanese hospital where he suddenly deceased after brief time. Clinical records, autopsy examination, histological findings, and toxicological analysis, assessed via a Q-Exactive Orbitrap with a HPLC system and LC/MS-MS system, are discussed. This case report may represent the first case of this type of intoxication and might help in improving recognition and treatment of these particular cases.

Detecting drugs in dry bone: a pilot study of skeletal remains with a post-mortem interval over 23 years.

In decomposed or skeletonized bodies, conventional matrices used in forensic toxicology may no longer be available for analysis. The aim of this paper was to test the survival and detection of toxicological substances in dry bone samples with over 23 years of post-mortem interval. In this perspective, bone samples from the cranium, ribs, and vertebrae of seven skeletons from the CAL Milano Cemetery Skeletal Collection, buried for over 23 years, fully decomposed and altered by taphonomic factors were selected based on their ante-mortem data, which included verified or suspected drug addictions or overdose. Qualitative and quantitative analyses were performed with Dionex™ ASE™ 350 Accelerated Solvent Extractor and Q-Exactive Orbitrap-mass spectrometry with a HPLC system. Positive results were obtained in six of the seven cases, and different psychoactive drugs (and in some cases their active metabolites) were detected, including analgesic (two opioids: methadone and buprenorphine) and anxiolytic drugs (benzodiazepines, in particular delorazepam, diazepam, nordiazepam, and lorazepam), a cannabinoid metabolite (THCCOOH) as well as metabolites of stimulants (benzoylecgonine and MDA). Consequently, this research shows that toxicological substances may be found in bone tissue after over 23 years of post-mortem interval.

Postmortem diagnostics of assumed suicidal food anaphylaxis in prison: a unique case of anaphylactic death due to peach ingestion.

Suicidal ingestion of food which the victim is aware they are allergic to is an exceptional occurrence in the forensic field. To the best of our knowledge, no cases of suicidal food anaphylaxis have been reported to date. Therefore we present the first case described in the literature. A 30-year-old prisoner was found dead inside his cell with the remains of a peach remains next to his body, and a handwritten farewell note in his pocket. The autopsy revealed only non-specific findings, while laboratory investigations (serological, toxicological, histological, and immunohistochemical) played a pivotal role in determing the cause and manner of death. In particular, a high titer of both total and specific IgE antibodies was detected, as well as an increase of the tryptase level in cadaveric blood. Moreover, a massive concentration of salicylates was measured in the gastric contents. Microscopically, cellular residues characterized by a vegetal structure were observed in the gastric contents and elements suggestive of mast cells were detected in the glottis, lungs, and myocardium. The immunohistochemical investigation with anti-CD117 and anti-tryptase antibodies showed positivity for mast cells, some of which appeared degranulated. Such findings were entirely consistent with an acute systemic anaphylactic reaction triggered by allergy. Therefore, the prisoner's death was attributed to self-induced food anaphylaxis caused by the ingestion of peaches. This conclusion was achieved based only on circumstantial data, anamnestic information, autopsy findings, and multiple laboratory results. This integrated approach should be used to pursue a post-mortem diagnosis of anaphylaxis.

Toxicological analysis in victims of sexual and domestic violence: A retrospective study of a 3-year period (2018-2020).

This manuscript presents an epidemiological investigation carried out on abuse victims who accessed the Sexual and Domestic Violence Service (SVS&D) of IRCCS Ca' Granda in Milan, Italy. The focal point of this research was the detection of alcohol, prescription medications, and illicit substances in victims who solicited help from the SVS&D center between 2018 and 2020. Over this three-year span, biological samples of blood and urine were procured from 207 victims, out of a patient pool of 2470. All collected samples were analyzed via High Performance Liquid Chromatography - Tandem Mass Spectrometry (HPLC-MS/MS) and Gas Chromatography - Mass Spectrometry (GC-MS). Toxicological examination results demonstrated that 43% of the cases tested positive for substances in 2018, 39% in 2019 and 60% of the cases in 2020. Overall, 45% of the victims tested resulted positive to some substance over a 3-year period, equivalent to 3.6% of the overall cases (2470 victims). Substances of toxicological interest were detected in 104 samples (out of 377, corresponding to 27.6%) belonging to 94 patients. The most detected classes of drugs were stimulants, antidepressants, benzodiazepines and antipsychotics. Moreover, BAC (Blood Alcohol Concentration) indicated positivity in 25 cases (out of 184 cases analyzed - 14% of positive cases). Based on this study's findings, we recommend broadening the range of substances evaluated in drug-facilitated sexual assaults and establishing standardized protocols for both national and international implementation. Implementing procedures would significantly enhance forensic support provided to victims of abuse seeking healthcare services post-incident.

Work-related and non-work-related fatal road accidents: Assessment of psychoactive substance use in commuting.

Commuting road traffic collisions (RTCs) are one of the main causes of occupational death worldwide, including Italy. Among the prominent yet relatively understudied personal risk factors in the subpopulation of workers, there is the use of psychoactive substances. Since the phenomenon of driving under alcohol and drugs effects in the commuter sub-population has so far been poorly studied, we carried out a study by comparing results obtained from the analysis of commuters and non-commuters. The percentage of commuting RTCs victims was 10.4% out of all the RTCs. Commuter victims were mostly men, 51-60 years-old, motorcyclists (32.1%) or car drivers (28.6%), employees, deceased during Fridays and Saturdays, in the afternoon (35.7%) and in the evening (32.1%). It was possible to perform toxicological analyses in 60.7% of commuter cases: approximately 40% tested positive, always and only for ethanol (median Blood Alcohol Concentration, BAC, of about 1.03 g/L). Values above the legal limit were observed in 23.5% of the toxicological samples analyzed from commuter accidents. Our findings contribute to bridging the gap in knowledge in the area of RTCs and commuting and emphasize the importance of carrying out toxicological investigations, with possible repercussions on both insurance issues and public health strategies, which are relevant for analyzing the phenomenon structurally.

Papaver somniferum in seventeenth century (Italy): archaeotoxicological study on brain and bone samples in patients from a hospital in Milan.

In this paper, we present the results of toxicological analyses of preserved brain tissue and bone samples from the remains of the seventeenth century patients of the Ospedale Maggiore, the main hospital in Milan and one of the most innovative hospitals in Europe from the Renaissance period. Beneath it, the crypt functioned as the burial place for the deceased of the hospital. In this multidisciplinary study of the remains, toxicological analyses in particular were performed with HPLC-MS/MS on different biological samples from nine individuals. Anthropological, paleopathological, histological, radiological examinations and radiocarbon dating were also carried out. As a result, archeotoxicological analyses revealed the presence of codeine, morphine, noscapine and papaverine, derived from Papaver somniferum, a plant present in the hospital pharmacopeia used as a narcotic, analgesic, astringent, coagulant, and antitussive agent. Such analyses have shed light on the pharmacological therapies administered to the patients near the time of death and have implemented our knowledge of medical treatment and drug administration in the 1600's.

Pharmacokinetics study about topical clobetasol on oral mucosa.

BACKGROUND: Clobetasol is the most potent topical corticosteroid used in oral medicine for muco-cutaneous diseases. Several papers reported about patients with cushingoid appearance, suggesting an adrenal suppression related to clobetasol systemic absorption after local application. Owing to the lack of studies, our goal is to assess whether transmucosal assimilation, after its application on oral mucosa, really occurs and to define clobetasol pharmacokinetics profile. METHODS: Data were recorded by collecting blood samples both on 10 patients in clobetasol therapy and on 14 healthy volunteers instructed about standardized clobetasol applications. A new technique of analytical chemistry was employed to detect its serum concentrations. RESULTS: Clobetasol absorption was ascertained, showing a certain accumulation rate. Different levels have been found in relation to oral disease and individual features (as smoking habits and presence of oral mucosa erosion). CONCLUSIONS: Our study validates clobetasol systemic transmucosal absorption, also recommending a careful monitoring of patients in corticosteroid therapy to avoid local and systemic adverse effects.

Postmortem forensic toxicology cases: A retrospective review from Milan, Italy.

We are presenting a study on 136 cases performed in a 2-year period (2018-2019) at the Bureau of Legal Medicine of the University of Milan for which toxicological analyses were requested and we are making a detailed interpretation of clinical records and discussing toxicological results from each case included in the study. Total number of autopsies was 1323 and in 10.3% of the cases, toxicological analyses were requested to obtain further information. Analyses were assessed with High-Performance Liquid Chromatography-Mass Spectrometry system and Gas Chromatography-Mass Spectrometry analyses. Additionally, Blood Alcohol Concentration and detection of volatile substances were obtained with Head Space-Gas Chromatography-Mass Spectrometry system. From these analyses, 101 cases out of 136 provided positive results (74.3%). Main substances detected were cocaine, diazepam, morphine, and ethanol. The most representative profiles of individuals that emerged from this study were: a Caucasian male, age 41-50, that died for cocaine acute intoxication or was killed; a Caucasian male or female with a range-of-age of 31-50 deceased for simple suicide caused by acute intoxication or by complex suicide caused by acute intoxication and suffocation; and finally, a Caucasian male with a range-of-age 21-40 that died in a car accident without any toxicological evidence. From the results, acute intoxication at the time of death was confirmed in 54 cases and in 57 cases the toxicological analyses helped in the determination of the cause and manner of death. From this study, the importance of toxicological data among forensic sciences is confirmed.

The Lethal Cutting: An Unexpected Cause of Death-A Methomyl Acute Intoxication.

Cocaine is among the illicit substances most frequently implicated in deaths related to the use of drugs of abuse both worldwide and in Italy. Cutting agents involved in the adulterations of this substance are many, and the process of lacing can take place at various stages of the production of the drug. In this report we are discussing the case of a 27-year-old woman found dead next to her car in a wooded area in the suburban area of Milan. On the crime scene, several specimens of white powder were collected and subsequently analyzed via Q-Exactive Orbitrap with an high performance liquid chromatography system and liquid chromatography--tandem mass spectrometry analysis along with biological matrices sampled during autopsy examination. The toxicological analysis revealed that the death could be ascribed to a lethal dose of methomyl, a carbamide pesticide used as cutting agent for cocaine. According to literature, this is the first time that this substance is used as an adulterant.

Histological paraffin-embedded block: a good alternative specimen to detect the use of opiates at least 20 years ago.

PURPOSE: Since the solely certain remnants of a performed autopsy are formalin-fixed paraffin-embedded (FFPE) samples, stored in the archives of every institute of legal medicine, we managed to extract molecules of toxicological interest from these specimens. METHODS: We assessed the analysis of ten fresh liver samples collected from heroin-related deaths and then histologically processed the same samples. The embedded blocks were then extracted by means of a new extracting method and the eluates were measured. We also selected five toxicological cases of heroin-related fatalities that were examined 20 years ago, collected the toxicological result documents of the analysis that were carried out at the time and then processed the corresponding FFPE liver samples that were stored in the archives. RESULTS: We managed to isolate heroine-related metabolites from 20-year-old paraffin-embedded blocks and calculated ratios to evaluate the performance of our new extraction. CONCLUSIONS: According to our study, it is feasible to carry out a toxicological examination on old histological samples and, therefore, this matrix can be considered as a new alternative specimen for chemical-analytical evaluations of past cases or when fresh samples are not available anymore. The new extractive method was evaluated as efficient in treating these complex, paraffin-embedded samples. It was surprising that the target compounds could be quantitated from FFPE bocks created as long as 20 years ago.

Driving under the influence of drugs: Correlation between blood psychoactive drug concentrations and cognitive impairment. A narrative review taking into account forensic issues.

Driving under the influence of alcohol has been shown to increase the risk of involvement in road traffic collisions (RTCs) however, less is known about the effects of illicit drugs, and a clear correlation between drug concentrations and RTC risk is still debated. The goal of this narrative review is to assess the current literature regarding the most detected psychoactive drugs in RTC (ethanol, amphetamines, cannabis, opioids and cocaine), in relation to driving performance. Evidence on impaired driving due to psychoactive substances, forensic issues relating to the assessment of the impact of drugs, blood cut-off values proposed to date as well as scientific basis for proposed legislative limits are discussed. At present there is no unequivocal evidence demonstrating a clear dose/concentration dependent impairment in many substances. Per se and zero tolerance approaches seem to have negative effect on drugged driving fatalities. However, the weight of these approaches needs further investigation.

How Do Drugs Affect the Skeleton? Implications for Forensic Anthropology.

Forensic anthropologists rely on a number of parameters when analyzing human skeletal remains to assist in the identification of the deceased, predominantly age-at-death, sex, stature, ancestry or population affinity, and any unique identifying features. During the examination of human remains, it is important to be aware that the skeletal features considered when applying anthropological methods may be influenced and modified by a number of factors, and particular to this article, prescription drugs (including medical and non-medical use) and other commonly used drugs. In view of this, this paper aims to review the medical, clinical and pharmacological literature to enable an assessment of those drug groups that as side effects have the potential to have an adverse effect on the skeleton, and explore whether or not they can influence the estimation of age-at-death, sex and other indicators of the biological profile. Moreover, it may be that the observation of certain alterations or inconsistencies in the skeleton may relate to the use of drugs or medication, and this in turn may help narrow down the list of missing persons to which a set of human remains could belong. The information gathered from the clinical and medical literature has been extracted with a forensic anthropological perspective and provides an awareness on how several drugs, such as opioids, cocaine, corticosteroids, non-steroidal anti-inflammatory drugs, alcohol, tobacco and others have notable effects on bone. Through different mechanisms, drugs can alter bone mineral density, causing osteopenia, osteoporosis, increase the risk of fractures, osteonecrosis, and oral changes. Not much has been written on the influence of drugs on the skeleton from the forensic anthropological practitioner perspective; and this review, in spite of its limitations and the requirement of further research, aims to investigate the current knowledge of the possible effects of both prescription and recreational drugs on bones, contributing to providing a better awareness in forensic anthropological practice and assisting in the identification process of the deceased.

An Optimized Terpene Profile for a New Medical Cannabis Oil.

The purpose of this analytical study was to develop an advanced formulation of medical Cannabis oil (MCO) comparing the chemical profile of different extracts obtained with two existing methods (SIFAP and CALVI) and one original upgraded (CERFIT) method. Preparation methods were applied with varying solvent, temperature, and duration of the decarboxylation and extraction steps. HPLC-MS/MS TSQ and GC/FID-HS analyses were performed to investigate cannabinoid and terpene contents in the three oil extracts. Cannabinoids profile remained comparable between the formulations. CERFIT extracts exhibited a superior quantity of total terpene hydrocarbon forms (e.g., limonene and α-pinene) with no degradation occurrence (i.e., oxidized terpenes not quantifiable). Thus, this new method optimized the phytochemical profile of the MCO presenting a value opportunity to obtain a standardized high-level therapeutic product.

Drugs in bone: Detectability of substances of toxicological interest in different states of preservation.

In forensic contexts of advanced decomposition, when conventional matrices are no longer available for toxicological analyses, finding alternative matrices is necessary. The skeleton, which is fundamental for anthropologists and geneticists, could be useful also for toxicological purposes. The present study aims to examine what kind of information toxicological analysis performed on bones (the cranium and the ribs) in different states of preservation could provide to the forensic practitioner. Thirty cadavers with known pharmacological history, subjected to forensic autopsy at the Institute of Legal Medicine of Milan, were selected. Rib and cranium samples were collected from each body and separated into two parts in order to create two different states of preservation: One was cleaned from soft tissues and analyzed as a well-preserved bone sample; the other was submitted to a long maceration process, simulating complete skeletonization. All specimens were then processed with accelerated solvent extraction and the eluates analyzed using Q-Exactive™ Orbitrap™ Mass Spectrometer. The analysis of blood and skeletal matrices showed positive results for the tested substances in 63% of cases, mainly benzodiazepines, antidepressants, and drug abuse. Significant Pearson correlations were observed between non-macerated vs. macerated bone samples: r = 0.79 for rib samples, r = 0.61 for cranium samples, and r = 0.69 for all bone samples. As a consequence, the positive results confirm the potential of the bone tissue as an alternative matrix in forensic toxicology, even in cases of extremely decomposed bodies. This study also highlighted important elements for reconstructing the biological profile in cases of forensic anthropological concern.

Thallium toxicity due to audultered infusion with thallium sulfate in eight members belonging to the same family nucleus: Autopsy findings and ICP-MS analysis (inductively coupled plasma mass spectrometry) in a triple homicide.

The banning of the heavy metal thallium (Tl) in many Countries, because of its toxicity, led to a remarkable reduction of the number of cases of poisoning both accidental and homicidal forcing us to better study the pharmacokinetics of this poison using new technologies. The Authors, in this work, are reporting the case of a collective thallium toxicosis caused by voluntary adulteration of an infusion with thallium sulfate, occurred in 8 members of the same familial nucleus; the administration of Prussian Blu resulted to be ineffective for 3 of these members that died at a later time. The most peculiar aspects of this rare manner of poisoning are discussed; the analytical procedures used, Inductively Coupled Plasma Mass Spectrometry (ICP-MS) in particular, resulted to be fundamental in the forensic diagnosis process of acute poisoning cause by thallium.

One Hundred and One Cases of Plastic Bag Suffocation in the Milan Area Between 1993 and 2013-Correlations, Circumstances, Pathological and Forensic Evidences and Literature Review.

Plastic Bag Suffocation is a rare cause of death in developed countries and almost unknown in the rest of the world. This study aims to retrospectively evaluate cases of PBS _asphyxia in Milan's Department of Legal Medicine from 1993 to 2013. Cases were selected from the database of 21,472 autopsies performed in the considered timeframe. One hundred and one cases were considered to be cases of Plastic Bag Suffocation, comprised of 100 suicides, no accidental events and 1 homicide. The most relevant elements pertaining to this type of death were evaluated for both the corpses and the crime scenes. From this study the typical PBS victim is an adult male, aged 52.3 years on average, depressed or afflicted by chronic or terminal diseases, and found at home. In 42.6% of cases, the victims were found with the plastic bag still positioned over their head, fastened by tying. Also common among these cases are drugs, alcohol and chemical abuses. According to the collected data, it is impossible to outline a pathognomonic detrimental background that leads to this cause of death. Crime scene investigation is, therefore, the one and only technical resource for evaluating these cases.

Predictive performance of the 'Minto' remifentanil pharmacokinetic parameter set in morbidly obese patients ensuing from a new method for calculating lean body mass.

BACKGROUND AND OBJECTIVES: In a previous article, we showed that the pharmacokinetic set of remifentanil used for target-controlled infusion (TCI) might be biased in obese patients because it incorporates flawed equations for the calculation of lean body mass (LBM), which is a covariate of several pharmacokinetic parameters in this set. The objectives of this study were to determine the predictive performance of the original pharmacokinetic set, which incorporates the James equation for LBM calculation, and to determine the predictive performance of the pharmacokinetic set when a new method to calculate LBM was used (the Janmahasatian equations). METHODS: This was an observational study with intraoperative observations and no follow-up. Fifteen morbidly obese inpatients scheduled for bariatric surgery were included in the study. The intervention included manually controlled continuous infusion of remifentanil during the surgery and analysis of arterial blood samples to determine the arterial remifentanil concentration, to be compared with concentrations predicted by either the unadjusted or the adjusted pharmacokinetic set. The statistical analysis included parametric and non-parametric tests on continuous variables and determination of the median performance error (MDPE), median absolute performance error (MDAPE), divergence and wobble. RESULTS: The median values (interquartile ranges) of the MDPE, MDAPE, divergence and wobble for the James equations during maintenance were -53.4% (-58.7% to -49.2%), 53.4% (49.0-58.7%), 3.3% (2.9-4.7%) and 1.4% h(-1) (1.1-2.5% h(-1)), respectively. The respective values for the Janmahasatian equations were -18.9% (-24.2% to -10.4%), 20.5% (13.3-24.8%), 2.6% (-0.7% to 4.5%) and 1.9% h(-1) (1.4-3.0% h(-1)). The performance (in terms of the MDPE and MDAPE) of the corrected pharmacokinetic set was better than that of the uncorrected one. The predictive performance of the original pharmacokinetic set is not clinically acceptable. Use of a corrected LBM value in morbidly obese patients corrects this pharmacokinetic set and allows its use in obese patients. The 'fictitious height' can be a valid alternative for use of TCI infusion of remifentanil in morbidly obese patients until commercially available infusion pumps and research software are updated and new LBM equations are implemented in their algorithms.