Laboratory of Environmental Hygiene and Forensic Toxicology

Poggio San Lorenzo, Italy

Laboratory of Environmental Hygiene and Forensic Toxicology

Poggio San Lorenzo, Italy

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Zamengo L.,Laboratory of Environmental Hygiene and Forensic Toxicology | Frison G.,Laboratory of Environmental Hygiene and Forensic Toxicology | Gregio M.,Laboratory of Environmental Hygiene and Forensic Toxicology | Orr G.,Laboratory of Environmental Hygiene and Forensic Toxicology | Sciarrone R.,Laboratory of Environmental Hygiene and Forensic Toxicology
Forensic Science International | Year: 2011

The determination of illicit active ingredients in seized materials, in order to assess penal or administrative offences, is routinely carried out in many forensic toxicology laboratories. This paper presents main features of the protocol adopted in the Authors' laboratory for the above investigations. In particular, sampling and analysis are considered as the same measurement process quantifying their combined contribution to overall measurement uncertainty. Aspects concerning representative sampling in the case of single and multiple items are discussed. The effects of material heterogeneity are considered by analyzing separately distinct primary samples taken from different parts of the sampling target. Possible errors due to particles dimension that could arise when sub-sampling are also considered. Analytical precision, bias and other matrix effects are studied in order to quantify the component of the overall measurement uncertainty associated to the analysis of prepared test samples. Typical scenarios arising when measurement results are used to assess compliance with specification limits are also discussed revealing the crucial role of measurement uncertainty. © 2010 Elsevier Ireland Ltd.


Frison G.,Laboratory of Environmental Hygiene and Forensic Toxicology | Odoardi S.,Catholic University of the Sacred Heart | Frasson S.,Laboratory of Environmental Hygiene and Forensic Toxicology | Sciarrone R.,Laboratory of Environmental Hygiene and Forensic Toxicology | And 4 more authors.
Rapid Communications in Mass Spectrometry | Year: 2015

Rationale We describe the analytical characterization of the designer drug bk-2C-B, a cathinone derivative, contained in a seized tablet, in the absence of an analytical standard. Methods The analytical techniques employed include gas chromatography/mass spectrometry (GC/MS), without and with derivatization with 2,2,2-trichloroethyl chloroformate, liquid chromatography/high-resolution-MS (LC/HRMS) with an Orbitrap® analyzer, and nuclear magnetic resonance (NMR). LC/HRMS measurements consisted of accurate mass measurements of MH+ ionic species under full scan conditions; comparison of experimental and calculated MH+ isotopic patterns; examination of the isotopic fine structure (IFS) of the M+1, M+2, M+3 isotopic peaks relative to the monoisotopic M+0 peak; study of MH+ collision-induced dissociation (CID) product ions obtained in fragmentation experiments. Results GC/MS analysis gave highly informative EI mass spectra, particularly after the derivatization of bk-2C-B with 2,2,2-trichloroethyl chloroformate. The application of LC/HRMS, allowing for accurate mass measurements at 100,000 resolving power, greatly enhanced analytical capabilities in structural characterization of this new designer drug. HRMS allowed us to obtain the accurate mass measurements of bk-2C-B MH+ ionic species, with a mass accuracy of 2.19 ppm; fully superimposable experimental and calculated MH+ isotopic patterns, with RIA1 and RIA2 values <4%; the IFS of the M+1, M+2, M+3 isotopic peaks relative to the monoisotopic M+0 peak completely in accordance with theoretical values. These findings enabled us to obtain the elemental composition formula of the seized drug. Furthermore, characteristic MH+ CID product ions enabled the characterization of the bk-2C-B molecular structure. The presence of 79Br and 81Br isotopes in the substance molecule produced a characteristic isotopic pattern in most MS spectra. Lastly, NMR spectra allowed us to obtain useful information about the position of substituents in the designer drug. Conclusions The combination of all the analytical techniques employed allowed the characterization of the seized psychoactive substance, in spite of the lack of a reference standard. Copyright © 2015 John Wiley & Sons, Ltd.


Frison G.,Laboratory of Environmental Hygiene and Forensic Toxicology | Zamengo L.,Laboratory of Environmental Hygiene and Forensic Toxicology | Zancanaro F.,Laboratory of Environmental Hygiene and Forensic Toxicology | Tisato F.,CNR Institute of Neuroscience | Traldi P.,CNR Institute of Neuroscience
Rapid Communications in Mass Spectrometry | Year: 2016

Rationale Clinical and forensic toxicology laboratories are challenged every day by the analytical aspects of the new psychoactive substances phenomenon. In this study we describe the analytical characterization of a new ketamine derivative, deschloroketamine (2-methylamino-2-phenylcyclohexanone), contained in seized powders. Methods The analytical techniques employed include gas chromatography/mass spectrometry (GC/MS), liquid chromatography/electrospray ionization coupled with Orbitrap high-resolution/MS (LC/ESI-HRMS), multistage MS (ESI-MSn), and NMR. The LC/ESI-HRMS analyses consisted of accurate mass measurements of MH+ ions in full-scan mode; comparison of experimental and calculated MH+ isotopic patterns; and examination of the isotopic fine structure (IFS) of the M + 1, M + 2, M + 3 isotopic peaks relative to the monoisotopic M + 0 peak. The collision-induced product ions of the MH+ ions were studied by both HRMS and MSn. 1H and 13C NMR measurements were carried out to confirm the chemical structure of the analyte. Results The EI mass spectra obtained by GC/MS analysis showed the presence of molecular ions at m/z 203, and main fragment ions at m/z 175, 174, 160, 147, 146, and 132. The application of LC/ESI-HRMS allowed us to obtain: the accurate mass of deschloroketamine MH+ ions with a mass accuracy of 1.47 ppm; fully superimposable experimental and calculated MH+ isotopic patterns, with a relative isotopic abundance value of 3.69 %; and the IFS of the M + 1, M + 2, M + 3 isotopic peaks completely in accordance with theoretical values. Examination of the product ions of MH+, as well as the study of both 1H and 13C NMR spectra, enabled the full characterization of the molecular structure of deschloroketamine. Conclusions The combination of the employed analytical techniques allowed the characterization of the seized psychoactive substance, in spite of the lack of a reference standard. Deschloroketamine is a ketamine analogue considered to be more potent and longer lasting than ketamine, and this paper is probably the first to report on its analytical characterization. Copyright © 2015 John Wiley & Sons, Ltd.


Zamengo L.,Laboratory of Environmental Hygiene and Forensic Toxicology | Bettin C.,Laboratory of Environmental Hygiene and Forensic Toxicology | Frison G.,Laboratory of Environmental Hygiene and Forensic Toxicology | Gregio M.,Laboratory of Environmental Hygiene and Forensic Toxicology | Sciarrone R.,Laboratory of Environmental Hygiene and Forensic Toxicology
Science and Justice | Year: 2013

Accurate and reliable analytical measurements are essential when data are to be used to assist the Court in deciding whether or not a drug offence has been committed and therefore about either the innocence or guilt of the accused. The Italian law on drugs demands that compliance with specification limits be assessed on the basis of the actual content of controlled substance contained in seized materials. As a consequence, the role of measurement uncertainty, significant figures and rounding errors becomes critical. In order to assist analysts of forensic toxicology laboratories with illicit drug-related cases, a software tool named Drugs WorkBook (DWB) has been developed. The tool is useful for the quantification of illicit drugs in seized materials along with their measurement uncertainties, the assessment of compliance to specification limits, the printing of comprehensive laboratory reports and the organization of case archives. Other quality control topics, such as control charts, are included. The tool's databases can be edited by the user and maintained up to date. The tool is made freely available to the scientific community. © 2013 Forensic Science Society.


Hair testing is considered to be one of the most efficient tool to investigate drug-related histories, particularly when the period of use needs to be tested back to many days or even months before sampling. High-resolution mass spectrometry represents today one of the most specific and sensitive analytical techniques to detect psychoactive substances in hair samples following single or multiple drug exposures. In this study pubic hair testing, by means of liquid chromatography-high resolution/high accuracy Orbitrap mass spectrometry, was employed to document the potential intake of five new psychoactive substances by a drug dealer. Pubic hair samples were decontaminated and pulverized with a ball mill, and, after the addition of the internal standard 3,4-methylenedioxypropylamphetamine, extracted with methanol:trifluoroacetic acid 9:1 at 45C for one night. The obtained extracts were analyzed on a Thermo Fisher Scientific Accela 1250 liquid chromatography system coupled to a Thermo Fisher Scientific single-stage Exactive HCD mass spectrometry system. 3-methylmethcathinone (3-MMC) was found to be present at a concentration of 25.8ng/mg in the pubic hair sample, whereas the other four designer drugs were found to be absent. 3-methylephedrines and 3-methylnorephedrines, metabolites of 3-MMC, were identified in the same sample, thereby proving the 3-MMC intake by the drug dealer.


PubMed | University of Rome La Sapienza, Catholic University of the Sacred Heart and Laboratory of Environmental Hygiene and Forensic Toxicology
Type: Journal Article | Journal: Rapid communications in mass spectrometry : RCM | Year: 2015

We describe the analytical characterization of the designer drug bk-2C-B, a cathinone derivative, contained in a seized tablet, in the absence of an analytical standard.The analytical techniques employed include gas chromatography/mass spectrometry (GC/MS), without and with derivatization with 2,2,2-trichloroethyl chloroformate, liquid chromatography/high-resolution-MS (LC/HRMS) with an Orbitrap analyzer, and nuclear magnetic resonance (NMR). LC/HRMS measurements consisted of accurate mass measurements of MH(+) ionic species under full scan conditions; comparison of experimental and calculated MH(+) isotopic patterns; examination of the isotopic fine structure (IFS) of the M+1, M+2, M+3 isotopic peaks relative to the monoisotopic M+0 peak; study of MH(+) collision-induced dissociation (CID) product ions obtained in fragmentation experiments.GC/MS analysis gave highly informative EI mass spectra, particularly after the derivatization of bk-2C-B with 2,2,2-trichloroethyl chloroformate. The application of LC/HRMS, allowing for accurate mass measurements at 100,000 resolving power, greatly enhanced analytical capabilities in structural characterization of this new designer drug. HRMS allowed us to obtain the accurate mass measurements of bk-2C-B MH(+) ionic species, with a mass accuracy of 2.19 ppm; fully superimposable experimental and calculated MH(+) isotopic patterns, with RIA1 and RIA2 values <4%; the IFS of the M+1, M+2, M+3 isotopic peaks relative to the monoisotopic M+0 peak completely in accordance with theoretical values. These findings enabled us to obtain the elemental composition formula of the seized drug. Furthermore, characteristic MH(+) CID product ions enabled the characterization of the bk-2C-B molecular structure. The presence of (79)Br and (81)Br isotopes in the substance molecule produced a characteristic isotopic pattern in most MS spectra. Lastly, NMR spectra allowed us to obtain useful information about the position of substituents in the designer drug.The combination of all the analytical techniques employed allowed the characterization of the seized psychoactive substance, in spite of the lack of a reference standard.


PubMed | Laboratory of Environmental Hygiene and Forensic Toxicology
Type: Journal Article | Journal: Science & justice : journal of the Forensic Science Society | Year: 2013

Accurate and reliable analytical measurements are essential when data are to be used to assist the Court in deciding whether or not a drug offence has been committed and therefore about either the innocence or guilt of the accused. The Italian law on drugs demands that compliance with specification limits be assessed on the basis of the actual content of controlled substance contained in seized materials. As a consequence, the role of measurement uncertainty, significant figures and rounding errors becomes critical. In order to assist analysts of forensic toxicology laboratories with illicit drug-related cases, a software tool named Drugs WorkBook (DWB) has been developed. The tool is useful for the quantification of illicit drugs in seized materials along with their measurement uncertainties, the assessment of compliance to specification limits, the printing of comprehensive laboratory reports and the organization of case archives. Other quality control topics, such as control charts, are included. The tools databases can be edited by the user and maintained up to date. The tool is made freely available to the scientific community.


Zamengo L.,Laboratory of Environmental Hygiene and Forensic Toxicology | Frison G.,Laboratory of Environmental Hygiene and Forensic Toxicology | Bettin C.,Laboratory of Environmental Hygiene and Forensic Toxicology | Sciarrone R.,Laboratory of Environmental Hygiene and Forensic Toxicology
Drug Testing and Analysis | Year: 2014

Cannabis is the most widely used illicit substance globally, with an estimated annual prevalence in 2010 of 2.6-5.0% of the adult population. Concerns have been expressed about increases in the potency of cannabis products. A high tetrahydrocannabinol (THC) content can increase anxiety, depression, and psychotic symptoms, and can increase the risk of dependence and adverse effects on the respiratory and cardiovascular systems in regular users. The aim of this study was to report statistical data about the potency of cannabis products seized in the north-east of Italy, in a geographical area centred in Venice and extending for more than 10 000km2 with a population of more than two million, by investigating the variability observed in THC levels of about 4000 samples of cannabis products analyzed over the period 2010-2012. Overall median THC content showed an increasing trend over the study period from about 6.0% to 8.1% (6.2-8.9% for cannabis resin, 5.1-7.6% for herbal cannabis). The variation in the THC content of individual samples was very large, ranging from 0.3% to 31% for cannabis resin and from 0.1 to 19% for herbal cannabis. Median CBN:THC ratios showed a slightly decreasing trend over the study period, from 0.09 (2010) to 0.03 (2012), suggesting an increasing freshness of submitted materials. Median CBD:THC ratios also showed a decreasing trend over the study from about 0.52 (2010) to 0.18 (2012), likely due to the increase in submissions of materials from indoor and domestic cultivation with improved breeding methods. © 2013 John Wiley & Sons, Ltd.


Strano Rossi S.,Catholic University of the Sacred Heart | Anzillotti L.,Catholic University of the Sacred Heart | Castrignano E.,Catholic University of the Sacred Heart | Frison G.,Laboratory of Environmental Hygiene and Forensic Toxicology | And 2 more authors.
Drug Testing and Analysis | Year: 2014

Zolpidem and zopiclone (Z-compounds) are non-benzodiazepine hypnotics of new generation that can be used in drug-facilitated sexual assault (DFSA). Their determination in biological fluids, mainly urine, is of primary importance; nevertheless, although they are excreted almost entirely as metabolites, available methods deal mainly with the determination of the unmetabolized drug. This paper describes a method for the determination in urine of Z-compounds and their metabolites by ultra-high-pressure liquid chromatography/tandem mass spectrometry (UHPLC-MS/MS) and UHPLC coupled with high resolution/high accuracy Orbitrap® mass spectrometry (UHPLC-HRMS). The metabolic profile was studied on real samples collected from subjects in therapy with zolpidem or zopiclone; the main urinary metabolites were identified and their MS behaviour studied by MS/MS and HRMS. Two carboxy- and three hydroxy- metabolites, that could be also detected by gas chromatography/mass spectrometry (GC-MS) as trimethylsylyl derivatives, have been identified for zolpidem. Also, at least one dihydroxilated metabolite was detected. As for zopiclone, the two main metabolites detected were N-demethyl and N-oxide zopiclone. For both substances, the unmetabolized compounds were excreted in low amounts in urine. In consideration of these data, a UHPLC-MS/MS method for the determination of Z-compounds and their main metabolites after isotopic dilution with deuterated analogues of zolpidem and zopiclone and direct injection of urine samples was set up. The proposed UHPLC-MS/MS method appears to be practically applicable for the analysis of urine samples in analytical and forensic toxicology cases, as well as in cases of suspected DFSA. © 2013 John Wiley & Sons, Ltd.


Zamengo L.,Laboratory of Environmental Hygiene and Forensic Toxicology | Frison G.,Laboratory of Environmental Hygiene and Forensic Toxicology | Tedeschi G.,Laboratory of Environmental Hygiene and Forensic Toxicology | Frasson S.,Laboratory of Environmental Hygiene and Forensic Toxicology | And 2 more authors.
Drug Testing and Analysis | Year: 2014

The measurement of blood-alcohol content (BAC) is a crucial analytical determination required to assess if an offence (e.g. driving under the influence of alcohol) has been committed. For various reasons, results of forensic alcohol analysis are often challenged by the defence. As a consequence, measurement uncertainty becomes a critical topic when assessing compliance with specification limits for forensic purposes. The aims of this study were: (1) to investigate major sources of variability for BAC determinations; (2) to estimate measurement uncertainty for routine BAC determinations; (3) to discuss the role of measurement uncertainty in compliance assessment; (4) to set decision rules for a multiple BAC threshold law, as provided in the Italian Highway Code; (5) to address the topic of the zero-alcohol limit from the forensic toxicology point of view; and (6) to discuss the role of significant figures and rounding errors on measurement uncertainty and compliance assessment. Measurement variability was investigated by the analysis of data collected from real cases and internal quality control. The contribution of both pre-analytical and analytical processes to measurement variability was considered. The resulting expanded measurement uncertainty was 8.0%. Decision rules for the multiple BAC threshold Italian law were set by adopting a guard-banding approach. 0.1g/L was chosen as cut-off level to assess compliance with the zero-alcohol limit. The role of significant figures and rounding errors in compliance assessment was discussed by providing examples which stressed the importance of these topics for forensic purposes. © 2014 John Wiley & Sons, Ltd.

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