Optimizing screening cutoffs for drugs of abuse in hair using immunoassay for forensic applications.

BACKGROUND: In forensic toxicology, positive immunoassay (IA) test results do not hold forensic validity and need to be confirmed with mass spectrometry (MS). On the other hand, a negative result is a strong indication that the drug and/or the drug metabolites are not present in the sample and that confirmatory analyses are not necessary. Consequently, a negative IA result must have forensic validity since it can be admitted in court during a trial. OBJECTIVES: Screening cutoffs for the analysis of hair samples using immunoassays (IAs) were retrospectively optimized based on the Society of Hair Testing (SoHT) confirmation cutoffs and the utility of the test for forensic applications was discussed. MATERIAL AND METHODS: Hair samples taken from 150 patients with a history of drug addiction were analyzed with ILab 650, Werfen (Milan, Italy) using DRI® reagents. Confirmatory analyses were subsequently performed using the ACQUITY UPLC® System, Waters Corporation (Milford, USA). Screening cutoffs were retrospectively optimized using receiver operating characteristic (ROC) analysis. RESULTS: A total of 162 single positive results were obtained for confirmatory analysis (10 for amphetamines/methamphetamines, 11 for MDMA, 37 for cocaine, 40 for THC, 33 for methadone, and 31 for opiates). The optimized screening cutoffs were 0.27 IA ng/mg for amphetamines, 0.51 IA ng/mg for MDMA, 0.59 IA ng/mg for cocaine, 0.14 IA ng/mg for cannabinoids, 0.63 IA ng/mg for methadone, and 0.26 IA ng/mg for opiates. An area under the curve (AUC) greater than 0.95 was obtained with very high sensitivity and specificity for all drugs. CONCLUSIONS: The presented screening method proved to be a useful technique on hair samples for the classes of drugs most commonly found in Italy and Europe and can be applied to forensic analysis.

Determination of ethyl glucuronide in hair and self-reported alcohol consumption in university students.

Young individuals constitute an intriguing population, as their drinking habits are notably shaped by their perception of their peers' alcohol consumption. Nonetheless, excessive alcohol intake can have detrimental effects on academic performance, interpersonal relationships, and the risk and severity of accidents. This study reported the first data involving students enrolled from three universities on a voluntary basis for alcohol consumption evaluation. Alcohol consumption was assessed through questionnaires and EtG quantification in hair (hEtG) carried out by liquid chromatography-mass spectrometry (LC-MS/MS) analysis after a solid-phase extraction (SPE) purification step. The results of our study demonstrated that 77.1% of samples tested negative for hEtG or displayed hEtG ≤ 5 pg/mg. Particularly, the student population was not characterized by samples with hEtG indicative of chronic excessive consumption (hEtG ≥ 30 pg/mg). No significant association was identified between biological sex, among the degree course/the year attended, nor in relation to BMI or smoking/coffee consumption. Among the obtained results, it was worth noting that the comparison of self-reporting abstinence from tobacco and coffee accounted for 65.3% and 16.7%, respectively, while only 2.8% of the total declared abstinence from alcohol. The current study has uncovered a significant level of interest among students in this analysis and its interpretation. This suggests that implementing public health promotion activities within a university setting could be beneficial.

Analysis and distribution of per- and polyfluoroalkyl substances in decidua and villi placenta explants.

Placenta mediates the transfer of nutrients, oxygen and drugs from mother to fetus. It is constituted by two cellular layers separated by the intervillous space: the outer is in direct contact with maternal blood (decidua placenta), and the inner (villi) directly in contact with the fetus. Environmental contaminants, such as per- and polyfluoroalkyl substances (PFAS) also demonstrated the ability to cross the tissue multiple layers, posing at risk the health of the fetus. The aim of the present study was to analyse the PFAS amount in decidua and villi placenta explants and to study differences in their distribution among the two side of this organ. The determination of 23 PFAS was carried out by liquid chromatography coupled to high-resolution accurate mass spectrometry (LC-HRAM). Our research included women who delivered at term between 2021 and 2022. Our data indicated that all samples contained at least one PFAS, demonstrating the ubiquitarian presence of these compounds in our population. A high occurrence of PFOS, PFOA and PFHxS, followed by PFHxA, PFBS and PFUnA was found. The fluorotelomer 6:2 FTS was also present in more than 40% of samples and this represent the first data on placenta explants. Mean and median PFAS values for decidual explants were 0.5 ng/g and 0.4 ng/g (SD 0.3), while for villi explants mean and median values were 0.6 ng/g and 0.4 ng/g (SD 0.4). A different pattern of accumulation was observed between villi and decidual explants for PFOS, PFOA and PFUnA (villi > decidua) and PFHxA, PFHxS, PFBS and 6:2 FTS (decidua > villi). Even if the mechanism of this selectively accumuation is not yet understood, molecular degree of ionization and its lipophilicity could at least in part explain this difference. This study expands the limited data describing PFAS levels in the placenta and pose attention on PFAS exposure during pregnancy.

Per- and polyfluoroalkyl substances (PFAS) presence in food: Comparison among fresh, frozen and ready-to-eat vegetables.

There is a worldwide discussion to provide safety limits in food for per- and polyfluoroalkyl substances (PFAS), a group of persistent contaminants associated to human disease. Processed food is more at risk of containing increased amounts of PFAS as a consequence of intentionally or non-intentionally contamination during manipulation and packaging. Among food products, also vegetables can be submitted to industrial manipulation; therefore, a different PFAS content correlated to the level of vegetables processing is conceivable. This study assessed the amount and type of PFAS present in fresh, frozen and ready-to-eat vegetables. Differences have been observed between the three groups of samples in the average PFAS content; the difference between ready-to eat and frozen vegetables resulted statistically significative. Organic vegetables displayed a lower total amount of PFAS respect to the traditional counterpart. The impact of industrial manipulation remains to be cleared, but pesticides use during cultivation could be considered a source of PFAS contamination.

Valproic acid determination by liquid chromatography coupled to mass spectrometry (LC-MS/MS) in whole blood for forensic purposes.

Valproic acid (VPA) is a well-known drug prescribed as anti-epileptic. It has a narrow therapeutic range and shows great individual differences in pharmacodynamics and pharmacokinetics. Consequently, the therapeutical drug monitoring (TDM) in patient's plasma is of crucial importance. Liquid chromatography coupled to mass spectrometry (LC-MS/MS) has gained importance in TDM applications for its features of sensitivity, selectivity and rapidity. However, in case of VPA, the LC-MS/MS selectivity could be hampered by the lack of a sufficient number of multiple reaction monitoring (MRM) transitions describing the molecule. In fact, the product ion scan of deprotonated molecules of VPA does not produce any ion and thus most LC-MS/MS methods are based on the detection of the unique MRM transition m/z 143➔143. In this way, the advantages of selectivity in LC-MS cannot be effectively exploited. In the present method, stable analyte adducts were exploited for the determination of VPA in blood. An Acquity HSS C18 column and mobile phases consisting of 5-mM ammonium formate and acetonitrile both added 0.1% formic acid were used. Source worked in negative acquisition mode and parameters were optimized to increase the adduct (m/z 189) and dimer (m/z 287) stability, and their fragmentation were used to increase the selectivity of MRM detection. The method has been validated according to the toxicological forensic guidelines and successfully applied to 10 real blood samples. Finally, the present method showed suitable for the rapid LC-MS/MS detection of VPA in whole blood, demonstrating the possibility to increase specificity by exploiting stable in-source adducts. This should be considered of utmost importance in the case of forensic applications.

Gender differences in driving under the influence of psychoactive drugs: Evidence mapping of real case studies and meta-analysis.

BACKGROUND: Very few studies have examined predictors of Driving Under the Influence (DUI) as a function of gender. This oversight is relevant, because analyzing gender differences prevents generalization of results observed in men, who still currently account for the majority of drivers worldwide, to women. The aim of this study is to analyze the prevalence of DUI of drugs in men and women reported in real case studies published in the last two decades, and to assess gender differences in risky DUI behaviour. METHODS: PubMed, Scopus, Web of Science were searched for eligible studies in May 2021; a follow-up literature search was conducted in August 2021. Real-case studies of drivers convicted for DUI of psychoactive drugs with positive toxicological confirmatory analysis were included. The extracted outcome was the prevalence of positive findings of men and women for cocaine, cannabinoids, amphetamine-like drugs, opioids, and psychoactive prescription drugs. A meta-analysis of random effects estimates was performed to investigate the change in the size of the overall effect (by Cohen d standardized mean difference test). A Mann Whitney U test was performed to test for differences between genders. RESULTS: Of the 2877 studies screened, 439 were retrieved in full-text and 26 were included. The meta-analysis showed a significant higher prevalence among men for cocaine (1.8% vs 0.9%; p < 0.001), cannabinoids (3.5% vs 1.6%; p = <0.01) and amphetamine-like drugs (1.2% vs 0.6%; p < 0.01). Surprisingly, no differences were observed in the use of opioids (2.3% vs 2.2%; p = 0.45) and benzodiazepines/Z-drugs (2.9% vs 3.7%; p = 0.52). CONCLUSION: Contrary to the extraordinary number of real-case studies reported in literature, only a few papers differentiate the prevalence of DUI between men and women. This can lead to an underestimation of the influence of gender in DUI phenomenon or complicate the evaluation of the results for some classes of substances, as observed for medications and opioids. The primary goal in the future will be to collect the data concerning DUI drivers following shared and homogeneous methodologies, in order to allow the analysis of data disaggregated by gender, which can be used for monitoring evolving trends and developing gender-specific targeted prevention and enforcement efforts.

Oral fluid analysis to monitor recent exposure to synthetic cannabinoids in a high-risk subpopulation.

Among novel psychoactive substances, synthetic cannabinoid receptor agonists (SCRA) seem to have the widest diffusion in the population with no limitation to any particular demographic group. Information on drug consumption relies mostly on anonymized surveys and less on clinical or analytical data; notwithstanding, the huge efforts constantly made to enroll subjects to gather epidemiological data. In the present study, we collected 66 oral fluid samples from volunteers in a drug rehabilitation center in 2019 and early 2020. A liquid chromatography-mass spectrometry method previously developed and validated by our Unit was applied to detect SCRA (n = 10) in oral fluid. Results proved the presence of synthetic cannabinoids at a positivity rate of almost 20%, with detection frequency HU211(5/13) > UR144/JWH122 (3/13) > JWH019/JWH081/AM2201 (1/13). Concentrations were in the range < LOQ -0.36 ng/ml. Synthetic cannabinoids consumption had not been declared by any volunteer. This study enabled for the unprecedent detection of synthetic cannabinoids use in the territory of Parma (Italy) in a high-risk subpopulation. The identified SCRA proved the persistence into the market of the "first-generation" JWH family into the Italian territory and the marketing of relatively new ones (AM-2201). Public health consequences represented by NPS consumption are still scarce; therefore, further studies are needed to understand the real diffusion in the population.

The use of fly artifacts in a crime scene: Is there any application for forensic toxicology?

Fly artifacts (FA) are bloodstains resulting from insect activity at a crime scene, usually by feeding on human blood. Whether these artifactual stains might be useful for forensic toxicological investigations in cases of absence of conventional and unconventional matrices, for example, in cases concealment of the body or of extensive putrefaction, has not yet been investigated. The purpose of this study is to understand if FA trace evidence permits toxicological analysis when traditional matrices are not available. To this aim, FA experimentally produced by Calliphora vomitoria feeding on human blood of a cocaine and heroin user were collected from absorptive and non-absorptive material. FA material was analyzed by a new simple and fast LC-MS/MS method. Results were evaluated in terms of presence of the drug and relative amount of the detected molecules. From a qualitative point of view, the analysis of FA revealed all the substances originally detected in post-mortem blood in both cases. The ratios of cocaine/benzoylecgonine, codeine/morphine, and 6-monoacetylmorphine/morphine recovered in FA from cotton-textile materials and from non-absorptive surfaces were consistent with data resulted from original post-mortem blood. The preliminary study herein reported demonstrated that FA are extremely informative in case of cocaine and heroin users and merit further research in order to be applied in real caseworks.

Molecular Mechanisms of Action of Novel Psychoactive Substances (NPS). A New Threat for Young Drug Users with Forensic-Toxicological Implications.

Novel psychoactive substances (NPS) represent a severe health risk for drug users. Even though the phenomenon has been growing since the early 2000s, the mechanisms of action of NPS at the receptors and beyond them are still scarcely understood. The aim of the present study was to provide a systematic review of the updated knowledge regarding the molecular mechanisms underlying the toxicity of synthetic opioids, cannabinoids, cathinones, and stimulants. The study was conducted on the PubMed database. Study eligibility criteria included relevance to the topic, English language, and time of publication (2010-2020). A combined Mesh and free-text protocols search was performed. Study selection was performed on the title/abstract and, in doubtful cases, on the full texts of papers. Of the 580 records identified through PubMed searching and reference checking, 307 were excluded by title/abstract and 78 additional papers were excluded after full-text reading, leaving a total of 155 included papers. Molecular mechanisms of synthetic opioids, synthetic cannabinoids, stimulants, psychedelics, and hallucinogens were reviewed and mostly involved both a receptor-mediated and non-receptor mediated cellular modulation with multiple neurotransmitters interactions. The molecular mechanisms underlying the action of NPS are more complex than expected, with a wide range of overlap among activated receptors and neurotransmitter systems. The peculiar action profile of single compounds does not necessarily reflect that of the structural class to which they belong, accounting for possible unexpected toxic reactions.

Automated sample preparation and analysis by gas chromatography tandem mass spectrometry (GC-MS/MS) for the determination of 3- and 2-monochloropropanediol (MCPD) esters and glycidol esters in edible oils.

The potentially carcinogenic process contaminant 3- and 2-monochloropropanediol esters (2-MCPD and 3-MCPD esters) and glycidyl esters (GEs) are under study in refined oils and foodstuffs. Legislation set recommended total daily intake (TDI) for 3-MCPD of 0.8 µg/kg and as low as reasonably achievable (ALARA) for glycidol. Usually, the so far adopted method for the determination of these contaminants relay on numerous and time-consuming steps for sample preparation (AOCS methods) and on GC-MS detection. The obtained sensitivities and the number of processable samples are thus limited. In this optic, new reliable methods that allow for the fast and sensitive determination of these contaminants in edible oils may be considered an improvement of the overall strategy of tackling the problem. In this paper a new automated method for sample preparation and detection by GC-MS/MS is presented and validated. Data on sensitivity (LOD at 1.5, 2.2 and 3 ng/g for 3-MCPD, 2-MCPD, 3-MBPD (deriving from glycidol), respectively), linearity across low and high calibration ranges and precision showed to be fit-for-purposes. Finally, the methodology was applied to ten extra virgin oil samples and one sample of sunflower seeds oil.

Determination of perfluoroalkyl substances (PFAS) in human hair by liquid chromatography-high accurate mass spectrometry (LC-QTOF).

Biomonitoring of perfluoroalkyl substances (PFASs) in hair is conventionally achieved by SPE extraction and liquid chromatography-triple quadrupole analysis, with sensitivities in the range of ng/g. The aim of this study was to develop and validate a rapid method to detect 20 perfluoroalkyl substances (PFASs) in human hair from general populations by SPE purification and liquid-chromatography coupled to accurate mass measurement (LC-QTOF). The obtained sensitivities (LOQ), linearity and RSD accuracies were respectively in the range of 0.07-0.5 ng/g, 0.1 (or 0.2 or 0.5)-10 ng/g, 1-16%. To verify the applicability of the method, 11 hair samples from volunteers were tested. The detected PFAS were PFBA (range 0.24-14.6 ng/g), PFBS (0.496 ng/g), PFOA (range 0.08-0.178 ng/g) and PFOS (

The Red Swamp Crayfish Procambarus Clarkii (the Louisiana Crayfish) as a Particular Scavenger on a Human Corpse.

An 85-year-old man was found deceased floating in an irrigation ditch 18 days after his disappearance. During crime scene investigation, specimens of the red swamp crayfish Procambarus clarkii were found in proximity of the body. The feeding activity of these nonendemic crayfishes contributed to the formation of specific injuries on the body and in the production of a large substance defects inside the corpse. The aim of this paper was to illustrate the scavenging activity of P. clarkii on a human body and highlight the potential postmortem artefacts caused by this species. This is the first report on a real case of postmortem injuries produced by P. clarkii crayfishes on a submerged human body. So far, crustaceans are not considered useful for the estimation of the minimum postmortem interval. However, the important modifications on the corpses deriving from the activity of these animals should be kept in consideration.

Zwitterionic HILIC stationary phase as a valuable alternative in separative techniques: Application to the analysis of gamma-hydroxybutyric acid and its metabolite in hair.

In this work, the physical and chemical properties of a novel zwitterionic LC stationary phase are applied to the development, validation and application of a new fast and reliable method devoted to the analysis of GHB (gamma-hydroxybutyric acid) and its relatively new discovered glucuronide metabolite in hair. The obtained sensitivity, expressed as limit of detection (LOD) and quantification (LOQ), were 0.033 and 0.10 ng/mg for GHB and 0.11 and 0.37 ng/mg, for GHB-glucuronide respectively. Linearity was assessed between LOQ and 50 ng/mg for both compounds. GHB and GHB-glucuronide extraction from hair matrix was maintained simple and consisted in an acidified-solvent incubation. No samples purification was required before LC-MS/MS analysis. The method was finally applied to 65 real hair sample, 60 adults and 5 children below 2 years old. The obtained results highlighted that GHB concentrations were in the range 0.11-0.96 ng/mg (average 0.38 ±â€¯0.25 ng/mg) in 44 cases (68%) while in 21 samples GHB concentrations were in the range between LOD and LOQ (0.033-0.1 ng/mg). GHB-glucuronide was detected in few samples (n. 3) at levels below LOQ. The interest on these molecules relies on the fact that GHB is both a naturally occurring inhibitory neurotransmitter in the central nervous system and an illicit drug often experienced by victims of drug-facilitated sexual assault. GHB-glucuronide was firstly identified in urine by the group of Petersen in 2013 and, as per analogy to ethyl glucuronide, it was proposed as a longer biomarker for GHB intoxication.

New psychoactive substances: An actual problem or an overestimated phenomenon?

In last years, international and national Institutions have been completely focused on the new psychoactive substances (NPS) phenomenon. Many contrast policies have been planned in order to control their spread. Even scientific entities, such as our Forensic Toxicology Division, have spent time and resources for NPS identification in biological (from clinical and forensic caseworks) and non-biological (seized material) samples. Last reports show a low prevalence of NPS across the Europe and Italy, while the classical drugs are still the main cause of drug-related deaths. In particular, a worrisome datum is represented by the increasing number of deaths due to heroin. Seen these statistics, is the NPS phenomenon overestimated? Is the interest in classical drugs decreased? Were we diverted by NPS?

Psychotropic substance abuse and fitness to hold a driving license in Italy.

Objective: Driving under the influence (DUI) of psychotropic substances is a serious and widespread problem in road safety. All countries try to reduce the impact with legislative controls over the criteria to regain a driver's license after suspension. In many European countries there are mandatory clinical and toxicological examinations required before a license is regranted. In Italy, individuals convicted of driving under the influence of drugs and/or alcohol must undergo a mandatory medico-legal and forensic toxicological examination prior to regranting of a license. This article reports on the prevalence, trends, and implications of psychotropic substances detected in more than 5,000 subjects submitted to driving license reissuance in the period 2011-2016. Methods: The study involved taking a clinical history, medical examination, and toxicological analysis of both urine and hair samples. Results: There was no change in the prevalence of psychoactive substances in the period 2011-2016. Cocaine was found most often (60%), followed by cannabinoids (15%) and opiates (9%). Methadone and amphetamine stimulants accounted for less than 5% each. Benzodiazepines were present in 15% of samples throughout the period. Conclusion: Cocaine and cannabinoids were the most used substances in the analyzed population, alone and in combination. Benzodiazepines were the most commonly detected prescription medication, raising questions about prescribed medication and driving risk that are not addressed by current legislation.

Novel zwitterionic HILIC stationary phase for the determination of ethyl glucuronide in human hair by LC-MS/MS.

Some recent studies have described a shift from traditional reversed-phase to more hydrophilic LC chemistry for EtG determination in hair (hEtG). The reason relies on the poor retention of C8- and C18-based columns for polar compounds, even in presence of great amount of aqueous phase. This work presents the development, validation and application of a new hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-LC-MS/MS) method based on a novel zwitterionic stationary phase for the analysis of hEtG. The linearity was assessed in the range of 5-100 pg/mg hair, with a correlation coefficient of >0.99. The method was selective and sensitive, with a limit of detection (LOD) and limit of quantitation (LOQ) of 1.4 pg/mg and 4.5 pg/mg in hair, respectively. Suitable diagnostic sensitivity was achieved without the introduction of a sample purification step, or a post column solvent addition. The method was successfully applied to real hair samples after full validation. This method, based on a separation at neutral conditions, confirmed the optimum retention and thus selectivity for weak acids in zwitterionic HILIC columns.

Proactive drugs in DFSA cases: Toxicological findings in an eight-years study.

In case of drug-facilitated sexual assault (DFSA), the evidence is frequently anecdotal, with few investigations based on scientific evidences being carried out and thus most cases are diagnosed as an acute drug or alcohol intoxication. The reason may lay in the lack of specific knowledge by the victim on the possibility to retrospectively study the allegedly events and to the absence of standardized and shared protocols among health, forensic and police subjects. On this basis, in 2015 the Unit of Forensic Toxicology of University of Florence and the Sexual Assaults Centre in Hospital Careggi have fixed a common protocol to be applied in case of DFSA. The purpose of the study was to describe the results of the application of the shared protocol for toxicological findings among women seeking health care after sexual assault, and to assess the relationship with so-called proactive DFSA drugs. We conducted a study on female patients above 18 years of age consulting the Sexual Assault Centre between 2010 and July 2018. Among the 256 patients included, 37.1% was positive at least for a substance. Alcohol was the most detected substance (57 cases), followed by Cannabis (19 cases), cocaine (15 cases) and opiates/methadone (heroine: 5; morphine:1; methadone: 6); benzodiazepines and amphetamine were found in 13 and in 2 cases, respectively. Only case of gamma-hydroxybutyrate (GHB) consumption was observed while new psychoactive substances were not detected. Among the patients suspecting proactive DFSA, sedative drug findings, not explained by voluntary intake, were encountered.

Application of HRAM screening and LC-MS/MS confirmation of active pharmaceutical ingredient in "natural" herbal supplements.

The growing market of herbal remedies worldwide could pose severe problems to consumers' health due to the possible presence of potentially harmful, undeclared synthetic substances or analogues of prescription drugs. The present work shows a simple but effective approach to unequivocally identify synthetic anorectic compounds in allegedly 'natural' herbal extracts, by exploiting liquid chromatography/time of flight (Q-TOF LC/MS) technology coupled to liquid chromatography/triple quadrupole (LC-MS/MS) confirmation and quantitation. The procedure was applied to five tea herbal extracts and pills sold as coadjutant for weigh loss. The method exploited liquid-liquid sample extraction (LLE) and separation in a C18 (2.1mm×150mm, 1.8µm) column. QTOF acquisitions were carried out both in scan mode and all ion MS/MS mode and results were obtained after search against ad hoc prepared library. Sibutramine, 4-hydroxyamphetamine, caffeine and theophylline were preliminary identified samples. Confirmation and quantitation of the preliminary identified compounds were obtained in LC-MS/MS after preparation of appropriated standards. Sibutramine, caffeine and theophylline were finally confirmed and quantitate.