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.

Cocaine profiling: Implementation of a predictive model by ATR-FTIR coupled with chemometrics in forensic chemistry.

In this study, a strategy based on Infrared Spectroscopy with Fourier Transformed and Attenuated Total Reflectance associated with chemometrics (ATR-FTIR) is proposed to identify the chemical "fingerprint" of cocaine samples. To this end, standard mixtures of cocaine and cuttings at differents ratio were investigated in order to develop a multivariate classification model to simultaneously predict the composition of the samples and to obtain a profile of adulteration of cocaine seizures. In addition, the application of a Partial Least Squares (PLS) and Principal Component Regression (PCR) calibration approaches were found to be a useful tool to predict the content of cocaine, caffeine, procaine, lidocaine and phenacetin in drug seizures. The achieved results on real confiscated samples, in cooperation with the Italian Scientific Investigation Department (Carabinieri-RIS) of Rome, allow to consider ATR-FTIR followed to chemometrics as a promising forensic tool in such situations involving profile comparisons and supporting forensic investigations.

Multi-class analysis of new psychoactive substances and metabolites in hair by pressurized liquid extraction coupled to HPLC-HRMS.

In this paper, an analytical method has been developed and validated for the analysis of new psychoactive substances (NPS) and metabolites in hair samples. The method was based on pressurized liquid extraction (PLE) followed by solid-phase extraction (SPE) clean-up and high performance liquid chromatography-high resolution mass spectrometry (HPLC-HRMS) analysis. To evaluate extraction efficiency and the applicability of the method, hair samples were fortified by soaking in order to obtain a good surrogate for drug users' hair; the amount of incorporated drugs related to their lipophilicity, similarly to in vivo drug incorporation. To the best of our knowledge, this is the first method that allowed for the analysis of both cathinones (5) and synthetic cannabinoids (7) in hair with a single extraction procedure and chromatographic run. A phenethylamine (2C-T-4), 4- fluorophenylpiperazine and methoxetamine were also included showing that PLE coupled to SPE clean-up was suitable for a multi-class analysis of NPS in hair. In addition, the use of PLE significantly reduced hair analysis time: decontamination, incubation, clean-up, and liquid chromatography-mass spectrometry (LC-MS) analysis were carried out in approximately 45 min. The method was fully validated according to Scientific Working Group for Forensic Toxicology (SWGTOX) and Society of Hair Testing (SoHT) guidelines. Limit of quantification (LOQ) values ranged from 8 to 50 pg mg-1 for cathinones, phenetylamines and piperazines, and from 9 to 40 pg mg-1 for synthetic cannabinoids (10 pg mg-1 for methoxetamine). Matrix effects were below 15% for all the analytes, demonstrating the effectiveness of the clean-up step. Inaccuracy was lower than 9% in terms of bias. Copyright © 2016 John Wiley & Sons, Ltd.

Broad Screening and Identification of Novel Psychoactive Substances in Plasma by High-Performance Liquid Chromatography-High-Resolution Mass Spectrometry and Post-run Library Matching.

Drug abuse is today a growing global problem. Often the consumers are not aware about the type of substances they are using and the correlated risks. In recent years, new psychoactive substances (NPS) appeared in the illicit market. The presence of NPS, such as synthetic cathinones, cannabinoids and phenethylamines, which are known to be pharmacologically and toxicologically hazardous, has been frequently reported. The aim of this study was the development of a liquid chromatography-high-resolution mass spectrometry (LC-HRMS) method for a broad screening of NPS in plasma. Data acquisition was in MS/MS and full-scan modes and the method was validated for 25 NPS belonging to different chemical classes. Quantitative results have been obtained for these analytes with limits of quantification ranging from 0.03 to 0.4 ng/mL. The method was proven to be suitable for the screening of additional substances; to this aim, a post-run library matching was conducted for every sample with an in-house database containing over 300 NPS and known metabolites. The library may be constantly expanded with new drugs, in order to obtain a broad screening of NPS in biological matrices.

Field detection capability of immunochemical assays during criminal investigations involving the use of TNT.

The capability to collect timely information about the substances employed on-site at a crime scene is of fundamental importance during scientific investigations in crimes involving the use of explosives. TNT (2,4,6-trinitrotoluene) is one of the most employed explosives in the 20th century. Despite the growing use of improvised explosives, criminal use and access to TNT is not expected to decrease. Immunoassays are simple and selective analytical tests able to detect molecules and their immunoreactions can occur in portable formats for use on-site. This work demonstrates the application of three immunochemical assays capable of detecting TNT to typical forensic samples from experimental tests: an indirect competitive ELISA with chemiluminescent detection (CL-ELISA), a colorimetric lateral flow immunoassay (LFIA) based on colloidal gold nanoparticles label, and a chemiluminescent-LFIA (CL-LFIA). Under optimised working conditions, the LOD of the colorimetric LFIA and CL-LFIA were 1 µg mL(-1) and 0.05 µg mL(-1), respectively. The total analysis time for LFIAs was 15 min. ELISA proved to be a very effective laboratory approach, showing very good sensitivity (LOD of 0.4 ng mL(-1)) and good reproducibility (CV value about 7%). Samples tested included various materials involved in controlled explosions of improvised explosive devices (IEDs), as well as hand swabs collected after TNT handling tests. In the first group of tests, targets covered with six different materials (metal, plastic, cardboard, carpet fabric, wood and adhesive tape) were fixed on top of wooden poles (180 cm high). Samples of soil from the explosion area and different materials covering the targets were collected after each explosion and analysed. In the second group of tests, hand swabs were collected with and without hand washing after volunteers simulated the manipulation of small charges of TNT. The small amount of solution required for each assay allows for several analyses. Results of immunoassays confirmed that they were suitable to detect post-blast residues in soil and target materials and post transfer residues on hands, allowing further confirmation by more selective techniques. ELISA and LFIAs results obtained from the same solution were consistently in good agreement, and were confirmed by gas chromatography coupled to mass spectrometry (GC-MS). The reported immunoassays data demonstrates the suitability of LFIAs as on-site rapid and effective assays to detect TNT traces. The CL-ELISA proved useful in obtaining very sensitive detection in forensic investigations and testing, while CL-LFIA had performances in between LFIA and CL-ELISA.

Pressurized liquid extraction for the determination of cannabinoids and metabolites in hair: Detection of cut-off values by high performance liquid chromatography-high resolution tandem mass spectrometry.

Hair analysis has become a routine procedure in most forensic laboratories since this alternative matrix presents clear advantages over classical matrices; particularly wider time window, non-invasive sampling and good stability of the analytes over time. There are, however, some major challenges for the analysis of cannabinoids in hair, mainly related to the low concentrations of 11-nor-9-carboxy-Δ(9)-tetrahydrocannabinol (THC-COOH), that is the major metabolite. In this study a fast, accurate and sensitive method for the determination of cannabinol, cannabidiol, THC and THC-COOH in hair has been developed. The extraction of analytes from hair (50mg) is based on an automated pressurized liquid extraction (PLE) using water modified with the surfactant sodium dodecyl sulphate as eluent phase. PLE extract is then cleaned up by SPE using polymeric reversed phase cartridges Strata XL before the injection in the HPLC-HRMS/MS system. Chromatographic conditions obtained with a fused-core column allowed a good separation of the analytes in less than 4min. The whole procedure has been validated according to SWGTOX guidelines. The LLOQs obtained for THC-COOH and the other analytes were respectively 0.1 and 2pg/mg. To the best of our knowledge, this is the first LC-MS/MS based method that allows the detection of THC-COOH in hair at values lower than the cut-off.

Forensic interlaboratory evaluation of the ForFLUID kit for vaginal fluids identification.

Identification of vaginal fluids is an important step in the process of sexual assaults confirmation. Advances in both microbiology and molecular biology defined technical approaches allowing the discrimination of body fluids. These protocols are based on the identification of specific bacterial communities by microfloraDNA (mfDNA) amplification. A multiplex real time-PCR assay (ForFLUID kit) has been developed for identifying biological fluids and for discrimination among vaginal, oral and fecal samples. In order to test its efficacy and reliability of the assay in the identification of vaginal fluids, an interlaboratory evaluation has been performed on homogeneous vaginal swabs. All the involved laboratories were able to correctly recognize all the vaginal swabs, and no false positives were identified when the assay was applied on non-vaginal samples. The assay represents an useful molecular tool that can be easily adopted by forensic geneticists involved in vaginal fluid identification.

The discrimination potential of diffuse-reflectance ultraviolet-visible-near infrared spectrophotometry for the forensic analysis of paper.

The application of diffuse reflectance UV-VIS-NIR spectroscopy is proposed to differentiate 20 office paper samples, which had been deemed similar by a preliminary visual examination under several different lighting sources. The samples were firstly screened on the basis of the qualitative appearance of their spectra. A further discrimination was obtained by taking into account three parameters: the average reflectivity of the paper samples in the range 680-900nm, and the integrated intensity of the absorption peak in the UV range at 272nm and at 360nm. The homogeneity of these parameters on both sides of the paper sheets was assessed, detecting a very uniform distribution of the optical brighteners. A special focus was posed on the determination of the discriminative power, in order to give a quantitative parameter on the proposed procedure, which is important when reporting results to the Court. The remarkable achievement of differentiating all the examined samples was obtained by UV-VIS spectroscopy, a very less expensive technique which is readily available in practically all forensic laboratories.

Molecular identification of vaginal fluid by microbial signature.

The discrimination of body fluids in forensic examinations can play an important role in crime scene reconstruction. Conventional methods rely on the detection of antigens or enzymatic activity, limiting detection sensitivity and specificity, particularly on old forensic samples. Methods based on human RNA analysis are not easily applicable to samples exposed to harsh and degrading environments. An alternative approach based on the identification of prokaryotic genomes was developed. Specific bacterial communities are characteristic typical of different human non-sterile body fluids: the molecular characterization of a microbial signature, and not the typing of single bacterial species, can effectively lead to univocal identification of these fluids. A multiplex real time PCR assay was developed using oligonucleotide mixtures targeting genomes specific for a selected group of bacteria. Microflora DNA (mfDNA) was extracted from vaginal, oral and fecal clinical swabs. In addition forensic samples were processed. Vaginal samples showed a strong specific signal for bacteria of the female genital tract. Oral samples clearly showed signal for bacteria present in saliva, and in fecal samples the main signal was from Enterococcaceae. Vaginal casework samples showed results comparable to freshly collected ones; moreover the DNA extracted was successfully used for STR typing. Also mixtures of body fluids were analyzed, providing a microbiological signature compatible with the presence of microbes of oral, fecal and vaginal origin. The presented method can be useful in identifying biological fluids, and it is based on DNA technologies already available in forensic laboratories and feasible for further high throughput automation.

Forensic differentiation of paper by X-ray diffraction and infrared spectroscopy.

The possibility to discriminate between sheets of paper can be of considerable importance in questioned document examinations. 19 similar types of office paper were characterized by infrared spectroscopy and X-ray diffraction to individuate the most discriminating features that could be measured by these techniques. The discriminating value associated to them was also assessed. By using a sequence of these two techniques, all the samples could be differentiated.