Microplastics and Nanoplastics in human tissues: Systematic review of evidence, analytical protocols, and methodological challenges Simone Ottavio Zielli, Jennifer Paola Pascali, Antonio Mazzotti, Paolo Fais, Milena Fini, Cesare Faldini, Susi Pelotti Talanta Open, 2026 Microplastics and NanoPlastics (MNP) have emerged as ubiquitous environmental contaminants with potential implications for human health. This systematic review synthesizes current evidence on the occurrence of MNP in human solid tissues and critically evaluates the analytical protocols employed for their detection and quantification. A comprehensive literature search conducted in September 2025 across MEDLINE, EMBASE, and the Cochrane Library, in accordance with PRISMA guidelines, identified 26 eligible studies encompassing 564 human samples from diverse biological matrices, including placenta, lung, liver, blood, and bone. Polyethylene, polypropylene, and polyvinyl chloride were the most frequently detected polymers, while particle sizes predominantly ranged between 20 and 100 µm. Analytical techniques varied substantially across studies, with Raman and Fourier-transform infrared (FTIR) spectroscopy representing the most widely applied methods, often complemented by microscopy or pyrolysis–GC/MS for polymer confirmation. Reported MNPs abundances ranged from less than one to several thousand particles per gram of tissue, reflecting the lack of standardized procedures for extraction, quantification, and contamination control. Recent investigations have increasingly implemented plastic-free workflows and procedural blanks, leading to improved reliability and reduced overestimation of MNP burden. Nevertheless, persistent methodological heterogeneity continues to hinder cross-study comparability and the establishment of true human tissue loads. Preliminary correlations between MNP accumulation and clinical or pathological parameters have been observed, but causal links remain unconfirmed. This review highlights the urgent need for internationally harmonized protocols, rigorous contamination prevention, and standardized reporting to ensure reliable biomonitoring and clarify the potential health implications of microplastic exposure in humans.
Assessing the stability of drugs of abuse and pharmaceuticals in postmortem blood samples Guido Pelletti, Susan Mohamed, Rafael Boscolo-berto, Alessia Giampietro, Arianna Giorgetti, Jennifer Pascali, Jacopo Lenzi, Susi Pelotti Advances in Clinical and Experimental Medicine, 2026 BACKGROUND: Reliable toxicological analysis is crucial for accurate forensic and clinical interpretation; however, pre-analytical factors such as handling and storage can significantly alter drug concentrations in postmortem (PM) samples, potentially leading to misinterpretation. Postmortem degradation, influenced by enzymatic and microbial activity, can change drug levels, making it essential to understand drug stability in biological matrices. OBJECTIVES: This preliminary study investigates the long-term stability of drugs of abuse and psychoactive substances in PM blood samples from drug-related deaths stored at -20°C for 29 months. MATERIAL AND METHODS: Postmortem blood samples were analyzed using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) following the routine methodology currently in use in the forensic toxicology laboratory. Stability was assessed by measuring concentration changes between analyses performed shortly after sample collection and reanalyses conducted after 6-29 months. Regression analyses were used to relate percentage variation in concentration to elapsed time. RESULTS: A strong correlation was found between the percentage reduction in drug concentration and storage time for all tested molecules, including morphine, cocaine, methadone, ketamine, benzodiazepines, antidepressants, antipsychotics, and lidocaine. Regression curve analysis revealed a reduction in concentration beginning within the initial months, with high variability. CONCLUSIONS: The study highlights the significant impact of long-term storage on drug concentrations in PM blood, emphasizing the need for careful consideration of storage intervals when reanalysis of samples is requested for forensic purposes. The findings underscore the importance of understanding degradation patterns for the accurate interpretation of toxicological results in medicolegal investigations.
Direct Analysis of Per- and Polyfluoroalkyl Substances (PFAS) in Plasma by Liquid Chromatography Coupled to Mass Spectrometry (LC–MS) for Biomonitoring Purposes Arianna Fornasari, Paolo Fais, Annamaria De Luca, Giovanni Dal Lago, Simone Santelli, Guido Pelletti, Elena Piva, Jennifer Pascali Exposure and Health, 2026 A rapid and direct liquid chromatography–mass spectrometry (LC–MS) method was developed and validated for high-throughput biomonitoring of Per- and Polyfluoroalkyl Substances (PFAS) in human plasma. This approach streamlines the analytical workflow by replacing traditional, labor-intensive solid-phase extraction (SPE) with a simple protein-precipitation step. Twenty-three PFAS (PFBA, PFPeA, PFHxA, PFHpA, PFOA, PFNA, PFDA, PFUnA, PFDoA, HFPO-Da, PFMPA, PFMBA, PFBS, PFPeS, PFHpS, PFOS, ADONA, 4:2 FTS, 6:2 FTS, 8:2 FTS, 9Cl-PF3ONS, 11Cl-PF3OUdS, PFEESA) were monitored. Method detection limits (MDLs) and limits of quantification (LOQs) ranged from 0.01 to 0.39 ng/mL and from 0.04 to 1.2 ng/mL, respectively. Accuracy (bias) and precision (RSD) were below 16% and 10%, respectively, for all PFAS in both intra- and inter-day experiments. Recoveries ranged from 86 to 102%, and matrix effects were estimated as ion suppression below 30% for all analytes. The method was successfully applied to a cohort of 426 Italian citizens who participated on a voluntary basis. Twelve PFAS were detected, with PFOS and PFOA present in 95% of the samples. The mean and median sums of detected PFAS were 7.3 ng/mL (SD 4.6) and 6.2 ng/mL $$IQR4.4{-}9.2$$ , respectively, with a maximum total concentration of 35 ng/mL. PFOS isomers (linear + branched) contributed the most to the total PFAS burden in plasma, reaching a maximum concentration of 25 ng/mL. Overall, this method proved to be a reliable and efficient tool for large-scale biomonitoring studies and provides an important basis for investigating the long-term health implications of PFAS exposure.
A Robust Multistep Digestion Method for Microplastics Detection in Human Tissue by MicroRaman Analysis Jennifer P. Pascali, Lucio Litti, Arianna Fornasari, Arianna Giorgetti, Michele Pozzebon, Rossella Barone, Antonio Ragusa, Paolo Fais Drug Testing and Analysis, 2026 The presence of microplastics (MPs) in human tissues has raised growing concerns, necessitating robust protocols for their reliable extraction and analysis. This study systematically evaluated and optimized digestion protocols to efficiently process a variety of human tissues—placenta, lung, kidney, adipose tissue, muscle, spleen, liver, thyroid, and brain—while preserving the integrity of MP particles. Initial assessments employing single‐reagent protocols such as nitric acid (HNO 3 ), proteinase K enzymatic digestion, and Fenton oxidative digestion demonstrated limited effectiveness, due to incomplete tissue breakdown or formation of turbid digestates that hindered filtration. Building upon these results, combined digestion approaches were investigated to improve organic matter removal and facilitate filtration through fine pore‐size filters (0.2 μm). The optimized 3‐day protocol included an initial oxidative Fenton digestion followed by enzymatic digestion (proteinase K). The final step involved lipid removal through ethanol addition and sonication, resulting in clear digestates amenable to filtration. This protocol efficiently digested complex tissue matrices, reducing filter clogging at 1‐μm size pore and preserving various common MP polymers, including low‐density polyethylene (LDPE), polyethylene terephthalate (PET), polytetrafluoroethylene (PTFE), and polyamides (PA6 and PA12). Application of the optimized digestion allowed successful isolation and characterization of MPs using optical microscopy and Raman spectroscopy. The method showed improved reproducibility and reliability over single‐reagent protocols, making it suitable for comprehensive MP analysis in human tissues. The application of an efficient and robust protocol for tissue digestion may contribute to advance human exposure assessment and toxicological studies related to MP contamination.
Emergency Department presentations due to new psychoactive substances (NPS) and other illicit drugs: a clinical and toxicological study on recreational drug toxicity in Italy Arianna Giorgetti, Annamaria Venturi, Rossella Barone, Francesca Rossi, Filippo Pirani, Jennifer Paola Pascali, Fabrizio Giostra, Guido Pelletti International Journal of Legal Medicine, 2026 Although the use of new psychoactive substances (NPS) has increased worldwide, current drug monitoring systems in emergency departments (EDs) across several European countries including Italy, remain mainly focused on classical drugs of abuse. This study aimed to assess the prevalence of NPS, ketamine and other illicit drugs in patients presenting to ED in Bologna, Italy, collecting associated clinical and toxicological data. This observational study included patients presenting to the ED of the University-Hospital of Bologna (IRCCS Azienda Ospedaliero-Universitaria di Bologna, Italy), between March and November 2024, with suspected acute recreational drug toxicity. Patient demographics and clinical features were recorded, and blood samples were analyzed for NPS and other drugs, using a previously validated and updated liquid chromatography-tandem mass spectrometry (LC–MS/MS) method. Of the 110 patients enrolled, 67 (60.9%) tested positive for at least one drug. Cocaine was the most frequently detected substance (n = 50; 74.6% of positive cases). Twenty patients tested positive for ketamine or norketamine (25.4% of positive cases), with mean values of 55 ng/ml and 101 ng/ml, respectively. NPS were detected in 3 patients (4.5% of positive cases) and consisted of methylone in 2 cases (below the limit of quantification), alpha-PHP (below the limit of quantification) and 3,4-MD-alpha-PHP in one case (16 ng/ml). Our study reveals a high prevalence of ketamine use, and a predominance of stimulants, particularly synthetic cathinones, among NPS. The association of NPS with psychomotor agitation underscores the clinical importance of considering these substances in cases of severe agitation. These findings emphasize the evolving landscape of recreational drug use and the critical role of comprehensive toxicological screening in emergency settings.
Determination of hair cortisol by liquid chromatography coupled to mass spectrometry (LC-MS/MS) as biomarker of chronic stress and application to academic students Elena Giovannini, Francesca Rossi, Jacopo Lenzi, Eleonora Berretti, Simone Santelli, Assia Benkhalqui, Filippo Pirani, Luca Morini, Jennifer P. Pascali Clinica Chimica Acta, 2026 Hair cortisol concentration (HCC), a non-invasive biomarker reflecting long-term cortisol exposure, offers valuable insight into chronic stress, complementing established acute stress measures such as salivary or blood cortisol. At the moment, the most valuable use of hair cortisol bio-analysis is to study trends and associations within specific populations rather than establishing a single cut-off value for stress level assessment. In this frame, a rapid LC-MS/MS method for HCC determination has been developed and applied to measure chronic stress in university students with the aim to correlate the analytical results with the perceived stress during the preparation of the exams. A total of 100 students from different academic programs were recruited providing hair samples and data on academic and lifestyle stressors. The method has been validated in accordance with forensic toxicological guidelines ensuring high sensitivity (LOD 2 pg/mg; LOQ 5 pg/mg) and robust performance across selectivity, linearity, accuracy, and stability parameters. Method linearity was assessed in the range 5-50 pg/mg; accuracy and precision calculated on QC were always below 7 %; prepared samples were stable for 4 days at refrigerated temperature. HCC was detectable in 94 % of samples in the range 5--47.7 pg/mg. Students attending Law and Biology courses exhibited the highest mean HCC values. Dietary changes and smoking were associated with higher stress perception. Among academic stressors, balancing work and study, as well as difficulties in study organization, were linked to greater perceived stress. No statistically significant correlation was found between perceived stress and HCC, underscoring the complexity of chronic stress assessment and the value of combining subjective and physiological indicators.
Integrating forensic pathology and toxicology for in-depth analysis of performance-enhancing substance misuse: Insights into injury, death, and broader implications Maria Paola Bonasoni, Francesca Rossi, Elena Piva, Elena Giovannini, Arianna Fornasari, Paolo Fais, Jennifer P. Pascali Legal Medicine, 2025 The death of a non-professional bodybuilder with a history of performance-enhancing drug (PED) use was thoroughly investigated. At the decedent's residence, investigators found four boxes of methylprednisolone tablets, one galenic preparation of sibutramine, and two unlabelled galenic compounds. Blood and hair samples were collected during autopsy. Toxicological analysis revealed the presence of methylprednisolone (19 ng/mL), methandrostenolone (27 ng/mL), and sibutramine (29 ng/mL) in blood. Hair analysis confirmed sibutramine (80 pg/mg) and methandrostenolone (1.28 ng/mg). GC-MS analysis of the seized substances detected methylprednisolone, sibutramine, and methandrostenolone in varying concentrations, highlighting cross-contamination during preparation. Death was related to a right cerebral endoventricular hemorrhage secondary to a ruptured subarachnoid arteriovenous malformation (AVM). The hypertensive peaks associated with weightlifting likely contributed to rupture of the weakened AVM walls. Indeed, histopathological analysis revealed myxoid parietal degeneration which was related to chronic hypertension induced by prolonged use of anabolic-androgenic steroids (AAS) and sibutramine. Evidence suggested that sibutramine was consumed both from labelled products and inadvertently from unlabelled compounds, highlighting safety concerns regarding the unregulated and poorly labelled substances. This case underscores the significant health risks associated with PED use, particularly in non-professional contexts where anti-doping measures are absent.
Per- and polyfluoroalkyl substances (PFAS) in placental compartments: Histopathological and toxicological data integration in an Italian cohort Arianna Giorgetti, Arianna Fornasari, Maria Paola Bonasoni, Alice Ferretti, Anna Seidenari, Maria Sech, Elena Piva, Jennifer P. Pascali, Paolo Fais Environmental Research, 2025 Per- and polyfluoroalkyl substances (PFAS) are synthetic environmental contaminants with widespread industrial and consumer applications, characterized by strong chemical stability and environmental persistence. Recent studies have highlighted placental permeability to PFAS, though evidence of direct histopathological impairment remains limited. This study aimed to investigate potential associations between PFAS exposure and histopathological abnormalities in placental samples. A total of 23 at-term pregnant women were recruited from two hospitals in Italy as part of a multicenter study. Placental samples, divided into maternal (decidua) and fetal (villi) compartments, were analyzed for PFAS concentration and histopathological alterations. PFAS were detected in 95.7% of samples. The most frequently detected PFAS were PFOS (88%), followed by PFHxS (83%), PFOA (83%), PFBS (54%) and PFHxA (54%). Preliminary findings suggest variable PFAS concentrations among subjects, with histopathological examination revealing placental alterations of potential clinical relevance. The observed histopathological alterations, particularly in cases of malperfusion and angiogenesis changes, suggest that PFAS may contribute to placental dysfunction, potentially affecting pregnancy outcomes. In particular, it could be hypothesized that PFHxA could exert an adverse influence on placental angiogenesis, due to pre-placental hypoxia stimulating the angiogenesis and resulting in increased ramification and number of branches. While direct causative links remain to be fully elucidated, these results underscore the need for further investigations into PFAS-related placental effects and their implications for fetal development. - PFAS were detected in 95.7% of samples - PFOS (88%), PFHxS (83%), PFOA (83%), PFBS (54%) and PFHxA (54%) were frequently detected - PFBS concentration in decidua samples was higher in pathological than normal placentas - Distal villous hypoplasia was observed in samples a higher number of PFAS - Higher PFHxA levels in villi were observed with increased angiogenesis
Analysis of Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD) on hair after single and repeated short in vivo passive exposures to low- and high-Δ9-THC-cannabis Arianna Giorgetti, Susan Mohamed, Francesca Rossi, Simone Santelli, Filippo Pirani, Guido Pelletti, Jennifer Paola Pascali, Susi Pelotti Forensic Science International, 2025 Prolonged cannabis smoke exposure could give rise to detectable levels of cannabinoids in hair, complicating forensic hair analysis interpretation. Exposure to "light cannabis", i.e., products that are low in Δ9-tetrahydrocannabinol (Δ9-THC) and enriched in cannabidiol (CBD), can additionally lead to contamination, as shown in vitro. The aim of the present study was to assess whether detectable hair levels of Δ9-THC and CBD could arise in vivo from short, single and repeated passive exposure to cannabis and "light cannabis" and whether the two products could be distinguished. Four volunteers underwent weekly 15-minute exposures to low-Δ9-THC (0.5 %) cannabis smoke, delivered by a pump inside a car, over a month. After 1 month of washout, exposures were repeated with the same scheme with high-Δ9-THC (5 %). Hair and urines samples were collected after each exposure. Hair samples were tested, with and without a washing step (total n = 72), by liquid chromatography tandem mass spectrometry for Δ9-THC and CBD. Urines were tested for drug metabolites (LOD: 1.66 ng/ml). No accumulation of drugs over exposures was shown. Urines always tested negative. Washed hair samples were positive for CBD (mean 0.05 ng/mg) after exposure to low-Δ9-THC cannabis, and for Δ9-THC (mean 0.02 ng/mg) after exposure to high-Δ9-THC cannabis, with levels also typical of drug use. The two products could be easily distinguished. Our study showed that hair contamination could arise in vivo even after short single exposures to cannabis and "light cannabis", underlining the need for a careful interpretation of results of hair analysis in forensic toxicology.
Human phase-I metabolism of three synthetic cannabinoids bearing a cumyl moiety and a cyclobutyl methyl or norbornyl methyl tail: Cumyl-CBMEGACLONE, Cumyl-NBMEGACLONE, and Cumyl-NBMINACA Arianna Giorgetti, Pietro Brunetti, Belal Haschimi, Benedikt Pulver, Jennifer Paola Pascali, Jan Riedel, Volker Auwärter Drug Testing and Analysis, 2025 Synthetic cannabinoid receptor agonists (SCRAs) continue to show high prevalence on the new psychoactive substances drug market. Around 2019–2020, new SCRAs bearing a cumyl moiety emerged: Cumyl‐CBMEGACLONE and Cumyl‐NBMEGACLONE, carrying a cyclobutyl methyl (CBM) and a norbornyl methyl moiety (NBM) attached to the γ‐carbolinone core. These were followed by Cumyl‐NBMINACA, the indazole carboxamide analog of Cumyl‐NBMEGACLONE. The study aimed at evaluating the human phase‐I metabolism of these compounds and at identifying suitable urinary markers to prove their consumption. After enzymatic hydrolysis, 14 authentic urine samples (eight for Cumyl‐CBMEGACLONE, four for Cumyl‐NBMEGACLONE, and two for Cumyl‐NBMINACA) were analyzed by liquid chromatography–quadrupole time‐of‐flight mass spectrometry. Results were compared with in vitro metabolites generated by pooled human liver microsomes incubation. Fifteen human phase‐I metabolites were identified for Cumyl‐CBMEGACLONE, nine for Cumyl‐NBMEGACLONE, and thirteen for Cumyl‐NBMINACA. The main in vivo metabolites were built by monohydroxylation, dihydroxylation, or trihydroxylation. The following urinary biomarkers are suggested for detecting the consumption of the investigated SCRAs: products of monohydroxylation at the CBM and at the core for Cumyl‐CBMEGACLONE; two products of monohydroxylation at the norbonyl methyl tail for Cumyl‐NBMEGACLONE; and metabolites built by dihydroxylation at the NBM substructure and by an additional hydroxylation at the cumyl moiety for Cumyl‐NBMINACA.
Evaluation of driving fitness in relation with the use of illicit and psychotropic substances. Epidemiological study of cases from Verona Giornale Italiano Di Medicina Del Lavoro Ed Ergonomia, 2011
Direct analysis of bromide in human serum by capillary electrophoresis Jennifer P. Pascali, Maristella Trettene, Federica Bortolotti, Giorgia de Paoli, Rossella Gottardo, Franco Tagliaro Journal of Chromatography B Analytical Technologies in the Biomedical and Life Sciences, 2006
Carbohydrate-deficient transferrin (CDT), new marker of chronic alcoholic abuse and its determination by capillary zone electrophoresis Rivista Di Medicina Di Laboratorio, 2004
