Sabina Strano Rossi

An analytical approach to the forensic identification of different classes of new psychoactive substances (NPSs) in seized materials

RATIONALE New psychoactive substances (NPSs) are rapidly spreading worldwide, and forensic laboratories are often requested to identify new substances for which no reference standards or analytical data are available. This article describes an analytical approach that was adopted in Italy by a few collaborative centres of the Italian Early Warning System for Drugs, which has contributed many alerts for the identification of different classes of NPSs in the last 24 months. METHODS Seized crystals and powders were initially analysed via single quadrupole gas chromatography/mass spectrometry (GC/MS), followed by liquid chromatography/high-resolution mass spectrometry (LC/HRMS) in the positive electrospray ionisation (ESI) mode at 100,000 full width at half maximum resolution (FWHM) without fragmentation to elucidate the elemental compositions of unknown molecules. Different fragmentation voltages during LC/HRMS were applied to study the accurate masses of the obtained characteristic fragments. Nuclear magnetic resonance (NMR) analyses were performed to identify specific isomers when necessary. RESULTS Some interesting examples of unknown NPSs from seizures later identified in our laboratories are reported, with special focus on those cases where analytical standards were not available during analyses. These cases include cathinones, such as 3-methylmethcathinone (3-MMC), methylone, bk-MBDB (butylone), 4-methylethcathinone (4-MEC), flephedrone, methylenedioxypyrovalerone (MDPV) and pentedrone, methoxetamine, apinaca or AKB48, benzydamine, meta-chlorophenylpiperazine (m-CPP), 5-MeO-N,N-dialkyl tryptamines, such as 5-MeO-DALT and 5-MeOMIPT, benzofurans, such as 6-APB and 4-APB, and diphenidine (identified for the first time in Europe). CONCLUSIONS The identification of NPSs in confiscated materials was successfully achieved via GC/MS coupled with LC/HRMS and, in a few cases, NMR analyses. The availability of GC/MS libraries is of great assistance in the identification of new drugs. Alternatively, the study of characteristic molecule fragments combined with the determination of their accurate masses can be a useful approach to identify unknown samples not previously analysed.

Drug distribution in the head, axillary and pubic hair of chronic addicts

This study investigates the distribution of some drugs in hair samples taken from different parts of the body (head, pubis and axillae). Samples drawn from 15 subjects who died from drug overdose were analysed. The concentrations of the drugs detected in the biological fluids did not appear to be correlated with those present in hair. The highest drug levels were observed in pubic hair. The concentration differences observed in the various types of hair can hypothetically be ascribed to a likely incorporation of the drugs from the outside.

A GC-MS method for the determination of sildenafil, vardenafil and tadalafil and their metabolites in human urine

Sildenafil (SDF), vardenafil (VDF) and tadalafil (TDF) are phosphodiesterase type 5 enzyme inhibitors (PDE5Is), used in the treatment of erectile disorders and to improve breathing efficiency in pulmonary hypertension. The increasing incidence of their use among young athletes has drawn the attention of the anti-doping authorities to the possible abuse of PDE5Is by athletes due to their pharmacological activities. This paper describes a method for the determination in urine of PDE5Is and their metabolites by gas chromatography/mass spectrometry (GC/MS) after liquid/liquid extraction of the analytes from urine and derivatisation to obtain trimethylsilyl derivatives. The metabolic profile was studied on real samples collected from subjects taking PDE5Is (Viagra, Levitra or Cialis); the main urinary metabolites were identified and their MS fragmentation characterized. The sample pre-treatment and GC/MS conditions for the detection of the metabolites have been optimised. A method for their preliminary screening and subsequent confirmation is described that takes into account the general requirements of a routine doping analysis to be used for the screening of large numbers of samples. The main metabolites identified can be included in a general purpose screening method and all the metabolites in a more specific confirmation method. The method developed has been applied for the screening of PDE5Is in 5000 urine samples. Based on the obtained results, the proposed method appears to be of practical use in analytical and forensic toxicology, including doping analysis.

IMPROVED ENZYMATIC-HYDROLYSIS OF HAIR

An enzymatic hair extraction method is proposed for drug analysis. Pronase digestion of various aliquots of hair from a cocaine abuser was preceded by a 2-h incubation with a dithiothreitol solution. The extraction solution was tested to identify possible interferences in the radioimmunoassay and was compared with other hydrolysis methods to assess the results of extraction.

Cocaine found in a child’s hair due to environmental exposure?

We report a case of a 6-year-old boy who had been living with his parents, both cocaine smokers, and who was urgently admitted to hospital for general distress. Upon examination, cocaine and cocaine metabolites were detected in hair and urine samples. These toxicological findings most likely indicate that the child had passively consumed the drug when living in a heavily contaminated environment.

Prevalence of illicit drug use among the Italian athlete population with special attention on drugs of abuse: A 10-year review

Abstract The objective of this study was to assess the prevalence of illicit drugs use among young adults, in particular elite athletes. This study considers the data obtained from anti-doping analyses performed on nearly 100,000 urine samples from 2000 to 2009 by the World Anti-Doping Agency accredited Italian Anti-Doping Laboratory. The percentage of adverse analytical findings varies on a yearly basis, but it is in the range 1.0–1.8% (not considering atypical findings, such as an altered endogenous steroid profile). Among positive results, there is a high prevalence of stimulants and drugs of abuse. The drug of abuse found most frequently is the tetrahydrocannabinol (cannabis) metabolite, accounting for 0.2–0.4% of the total samples analysed (18% of the positive results). The second most frequently encountered drug is cocaine, as detected from cocaine metabolites, accounting for 0.1% of the total samples analysed (7% of positive results). Other stimulants found included amphetamines, ephedrines, carphedon, modafinil, and anorexic compounds. No amphetamine-like designer drugs were detected. These data are indicative of the widespread prevalence of cocaine and cannabis use among the young adult population. However, due to the particular population studied, it must be considered an underestimation of the phenomenon among elite athletes with respect to the general population.

Simultaneous detection of cocaine and heroin metabolites in urine by solid-phase extraction and gas chromatography-mass spectrometry

The present paper reports a method for the simultaneous extraction of cocaine, heroin and their metabolites from small amounts of urine (0.5 ml), using deuterated internal standards. Solid-phase extraction (SPE) on C18 columns followed by chromatographic separation coupled with mass spectrometry allowed the detection of all the substances after their derivatization. Mass spectrometry was performed in the electron-impact selected-ion monitoring (EI-SIM) mode. The limit of detection was found to be as low as 50 ng/ml for all the analytes; for reproducibility the C.V. was always better than 7%; the method was found to be linear with correlation coefficients between 0.989 and 1.00.

A fast gas chromatography/mass spectrometry method for the determination of stimulants and narcotics in urine.

A fast method has been developed for the simultaneous determination of 52 stimulants and narcotics excreted unconjugated in urine by gas chromatography/mass spectrometry (GC/MS). The procedure involves the liquid/liquid extraction of the analytes from urine at strong alkaline pH and the injection of the extract into a GC/MS instrument with a fast GC column (10 m x 0.18 mm i.d.); the short column allows the complete separation of the 52 analytes in a chromatographic run of 8 min. The method has been fully validated giving lower limits of detection (LLODs) satisfactory for its application to antidoping analysis as well as to forensic toxicology. The repeatability of the concentrations and the retention times are good both for intra- and for inter-day experiments (%CV of concentrations always lower than 15 and %CV of retention times lower than 0.6). In addition, the analytical bias is satisfactory (A% always >15%). The method proposed here would be particularly useful whenever there are time constraints and the analyses have to be completed in the shortest possible time.

Rapid and simple procedure for the determination of cathinones, amphetamine-like stimulants and other new psychoactive substances in blood and urine by GC–MS

Graphical abstract Figure. No caption available. HighlightsHigh‐throughput method for stimulant NPS determination in biological samples.Ultra‐rapid, simple, low solvent‐use, economic.GC–MS analysis in SCAN mode.Possible reprocessing of datafiles to uncover further analytes. ABSTRACT In the last few years an increasing number of new psychoactive substances (NPS), with different chemical structures (of which 37% are stimulants), have been released into the illicit drug market. Their detection and identification in biological samples is hence of great concern. The aim of this work was to develop a high‐throughput and rapid method for the determination of different classes of stimulants (amphetamine‐type stimulants, cathinones, phenethylamines and ketamine analogues) from blood and urine samples using GC–MS. The proposed method allows the almost simultaneous derivatization and extraction of analytes from biological samples in a very short time, by using hexyl chloroformate as derivatization agent. The extraction of analytes was performed by Dispersive Liquid Liquid Microextraction (DLLME), a very rapid, cheap and efficient extraction technique that employs microliter amounts of organic solvents. The chromatographic method allowed for the separation of 26 stimulants including positional isomers (3‐MMC and 4‐MMC). The method was validated on urine and blood samples with the ability to detect and quantify all analytes with satisfactory limits of detection (LODs) ranging between 1 and 10 ng/mL, limits of quantification (LOQs) between 2 and 50 ng/mL, selectivity and linearity (5–1000 ng/mL). The method was then applied to real samples from forensic cases, demonstrating its suitability for the screening of a wide number of stimulants in biological specimens.