Raffaele Giorgetti

Determination of lamotrigine in whole blood with on line solid phase extraction

A simple, sensitive and reproducible method was developed for the determination of lamotrigine in whole blood with on-line solid phase extraction followed by HPLC separation with UV detection. Whole blood samples were diluted 1:1 with water and then injected directly on a clean-up column dry-packed with 40microm C8 silica and separated on a C18 reversed-phase column (150x4.6mm) at room temperature. The extraction column was activated with methanol and conditioned with phosphate buffer of pH 4.5. Mobile phases consisted of phosphate buffer of pH 4.5 for the extraction column and of phosphate buffer of pH 4.5 - acetonitrile (60:40, v/v) for the analytical column. At a flow rate of 1.0ml/min and a connection time of 1.0min, the complete cycle time was 10.0min. Detection was carried out at 260nm. No internal standard was necessary. The method was linear over concentration range 0.2-20.0microg/ml for lamotrigine. Recovery was 98%. Within-day and between-day coefficients of variation ranged from 1.8 to 6.7%.

When “Chems” Meet Sex: A Rising Phenomenon Called “ChemSex”

Background: The term “chemsex” was coined to indicate the voluntary intake of psychoactive and non psychoactive drugs in the context of recreational settings to facilitate and/or to enhance sexual intercourses mostly among men who have sex with other men (MSM). Objective: The authors aimed to review the mechanisms of action, the toxicity and the pattern of use and abuse of substances involved in “chemsex” practice together with the sociocultural background underlying it and the health-related consequences that they may have. Results: Gamma-hydroxybutyrate, gamma-butyrolactone,1,4-butanediol, mephedrone, methamphetamine, sildenafil, tadalafil, vardenafil and alkyl nitrites have been described in their role of “chemsex drugs” including pharmacological action and in their implication to impair capacities to chose sexual partners and consensual sex. Moreover, it has been demonstrated that sexual activity over protracted length of time under the influence of chemsex drugs can result in rectal trauma or penile abrasions and a significant increase of the risk of transmission of sexual transmitted diseases, especially in case of condomless intercourses, which are frequent in this context, representing therefore a serious health threat. Conclusion: One of the major problems to establish health policy priority interventions for chemsex is the lack of available epidemiological data on the issue. Finally, social actions should be taken in order to break down the barriers that currently exist among chemsex drug users in accessing services, including the shame and stigma often associated with drug use. In conclusion, more specific resources to face high risks of infections and HIV transmission are required in bisexual and homosexual individuals having SUID: sex under the influence of drugs.

Suitability of PCR methods for forensic investigation. Analysis of the 3'apoB VNTR system in an Italian population sample.

SummaryThe PCR method has been applied to amplify a Variable Number Tandem Repeat (VNTR) sequence located at the 3′ end of the apolipoprotein B (ApoB) gene. The study was conducted on an Italian population sample and in a 3-generation family of 13 members, whose relationships were previously established using conventional blood systems. The allele frequencies found were compared with those reported in the literature. The results also confirmed the Mendelian inheritance of the alleles and the suitability of the PCR method for forensic purposes.ZusammenfassungDie PCR Methode wurde angewendet, um eine VNTR-Sequenz zu amplifizieren, welche am 3. Ende des Apolipoprotein B-Gens lokalisiert ist. Die Studie wurde an einer italienischen Populations-stichprobe durchgeführt und in einer 3-GenerationenFamilie mit 13 Mitgliedern, deren Verwandtschaftsverhältnisse vorher durch konventionelle Blutgruppensysteme abgesichert wer. Die gefundenen Allelfrequenzen wurden mit jenen aus der Literatur verglichen. Die Resultate bestätigen die Mendelsche Vererbung der Allele und die Anwendbarkeit der PCR-Methode für forensische Zwecke.

Morphometric Investigation of Death by Asphyxia

Abstract:  The aim of this study was to investigate the possibility of distinguishing deaths by asphyxia from those due to natural causes by comparing morphometric measurements in lungs. The study population comprised 27 subjects: 14 cases of death by asphyxia (hanging or drowning) and 13 cases of sudden natural death. Eighty parenchyma sections were used for each subject. Data were collected by computerized image analysis. Measurements aimed at quantifying, as percentages, pulmonary parenchyma (mean values of positive‐fraction areas [PFA] and their standard deviations). Subjects who died of natural causes and of drowning showed a relative internal homogeneity compared to those who hanged. Results show significant discrimination between drowned subjects and those dying of natural causes (mean of PFA p = 0.01) and between hanged and drowned subjects (SD of PFA p = 0.04). Attention must be paid to the possible role played by senile emphysema. The method is proposed as a complementary tool in forensic cases.

Fentanyl novel derivative-related deaths

Fentanyl (FEN) is a potent, synthetic narcotic used as an anaesthetic and a pain reliever, but also illegally manufactured. For diversion purpose, it is being steadily modified to produce new analogous compounds and derivatives (FENS), categorised as novel psychoactive substances. While potential FEN abuse is already known, even in the absence of a clear lethal dosage, there is still a shortage of data on its derivatives.

Stability of endogenous GHB in vitreous humor vs peripheral blood in dead bodies

For the first time, the stability of GHB was tested in post-mortem peripheral blood and vitreous humor samples, collected from 22 dead bodies at two different times: at the external body examination at the place of death and then during autopsy. An ad hoc method for the detection and quantification of GHB in vitreous humor by gas chromatography coupled to mass spectrometry (GC-MS) was developed and validated, with a good linearity between 0.1 and 50μg/mL (r2=0.991) and a precision and accuracy always better than 10% and an analytical recovery higher than 90%. The geometric mean of GHB concentration in the 22 peripheral blood samples at t0 was: 3.6μg/mL (95% CI: 2.3-5.9μg/mL) and at t1 it was 7.4μg/mL (95% CI: 5.0-10.9μg/mL); that of GHB in the 22 vitreous humor at t0 was: 2.5μg/mL (95% CI: 1.5-4.1μg/mL) and at t1 it was 3.0μg/mL (95% CI: 1.9-4.8μg/mL). There was no significant difference between the GHB concentrations in vitreous humor and peripheral blood at t0 in all the samples (p>0.10). Conversely at t1, the increase of GHB in the peripheral blood was significantly increased by a 102% (range: 86-120%) (p<0.001 vs t0), while in the vitreous humor only a slight increase by 19% was observed (range: 16-21%) (p>0.05 vs t0). Finally at t1, GHB values in the two matrices were statistically different, being that of peripheral blood higher (p<0.01). This study demonstrated the usefulness of vitreous humor as a more stable alternative matrix in comparison to peripheral blood for the post-mortem determination of endogenous GHB.

Effects of GHB on psychomotor and driving performance

BACKGROUND Gamma-hydroxybutyrate (γ-hydroxybutyrate or GHB) is a physiological compound of mammalians with specific receptors in central nervous system (CNS). Apart from an endogenous production, GHB is also an exogenous molecule found in pharmaceutical as well as in illicit formulations. Products manufactured and spread in these two fields, whose borders are all but strictly defined and traced, differ for the in vivo effects and for the potential of abuse. Illicit GHB gained public attention due to its use as a sexual assault facilitator. Notwithstanding its popularity, the effects on human performances, are still not completely understood and could be more complex than expected. Therefore, there is a real public safety concern regarding psychomotor functions and driving abilities due to GHB ingestion. OBJECTIVE To provide an updated and complete review on the effects of GHB on psychomotor, cognitive and driving performance that may be useful for judiciary expert forensic evaluation of driving under the influence of GHB (GHB-DUI). METHOD Experimental animal-based and human-based studies and case series or epidemiological studies regarding driving under influence of GHB (and its precursors) were reviewed using main scientific databases. RESULTS The effects of GHB on cognitive, psychomotor and driving performance are dose-related in experimental studies. In real cases of driving under the influence of GHB, severe impairment is observed. In these cases, a wide range of blood GHB levels are found. Possible long-term effects are also reported. CONCLUSION GHB causes cognitive and psychomotor impairment and risky driving behavior. Multiple aspects and variables are still waiting clarification, such as the harmful potential of illicit preparations, the effect of precursors and impairing dosages. GHB (and its precursors) must be considered a substantial personal and public risk even in the absence of a clear dose-effects correlation.

Investigation on genetic thrombophilic factors in FFPE autopsy tissue from subjects who died from pulmonary embolism

Venous thromboembolism (VTE) is a multifactorial disease determined by a combination of inherited and acquired factors. Inherited factors include mutations in the genes coding for coagulation factors, some of which seem to exert a differential influence on the risk of developing deep vein thrombosis (DVT) and pulmonary embolism (PE). In post-mortem studies of subjects who have died from pulmonary embolism (PE), the analysis of the factors that may have augmented the VTE risk is often limited to acquired factors. This is due to the complexity—and sometimes the unfeasibility—of analyzing genetic factors and to insufficient knowledge of their individual roles in PE development. The present study used formalin-fixed paraffin-embedded (FFPE) tissue to investigate a panel of 12 polymorphisms—the largest ever studied—that affect the VTE risk. Tissue samples came from post-mortem examinations performed by the specialists of the Section of Legal Medicine of the Department of Pathology of Marche’s Polytechnic University, and by the specialists of Health Care District Hospital of Imola, on 44 subjects who died from PE in the period 1997–2014. All individuals were found to have at least one mutation affecting the VTE risk. The present study demonstrates that genetic analysis can be performed post-mortem and the results are useful for forensic investigations, especially from MTHFR C677T and PAI-1 4G/5G polymorphisms. Broader studies using the techniques described herein are needed to determine the relative influence of the individual polymorphisms and their interaction in PE deaths.

Pharmacotoxicology of Non-fentanyl Derived New Synthetic Opioids

A class of opioid agonists not structurally related to fentanyl, derived from research publications of pharmaceutical companies or patents within the United States and abroad are contributing to the current opioid epidemic. Novel synthetic opioids (NSOs) created to circumvent drug control laws such as U-47700, U-49900, AH-7921, or MT-45 have no recognized therapeutic use, are clandestinely manufactured and sold on conventional or dark web. We herein provide a review of the pharmacological properties available on most of these substances trying to provide a better knowledge on these compounds, particularly with respect to toxicity and dangerous adverse effects in users. Indeed, these NSOs share not only a great potency of action and receptor affinity with respect to natural or synthetic opiates (e.g., morphine, heroin, and methadone) but also a non-negligible toxicity leading to intoxications and fatalities, posing a serious harm to public health and society.

Massive Lesions Owing to Motorcyclist Impact Against Guardrail Posts: Analysis of Two Cases and Safety Considerations*

Abstract:  Two motorcycle riders lost control of their vehicle, fell, and hit a guardrail, which acted as a blade and led to a rapid, fatal outcome. In one case, the high velocity of the body at the time of the impact resulted in complete detachment of the trunk. Reconstruction of the accident dynamics enabled the guardrail post to be identified as the means of injury in both cases. The two accidents occurred over a short period of time, highlighting a dangerous phenomenon that in less severe cases is presumably associated with different degrees of survivor disability. The accidents deserve mention, because a different design of the impact surface of the guardrail post might have prevented the lethal outcome. There is an urgent need for legislators to pass regulations that modify crash barrier homologation criteria, which have been devised primarily for the safety of car passengers but fail to protect motorcyclists.