Early detection of emerging street drugs by near infrared spectroscopy and chemometrics.

Near-infrared spectroscopy (NIRs) is spreading as the tool of choice for fast and non-destructive analysis and detection of different compounds in complex matrices. This paper investigated the feasibility of using near infrared (NIR) spectroscopy coupled to chemometrics calibration to detect new psychoactive substances in street samples. The capabilities of this approach in forensic chemistry were assessed in the determination of new molecules appeared in the illicit market and often claimed to contain "non-illegal" compounds, although exhibiting important psychoactive effects. The study focused on synthetic molecules belonging to the classes of synthetic cannabinoids and phenethylamines. The approach was validated comparing results with officials methods and has been successfully applied for "in site" determination of illicit drugs in confiscated real samples, in cooperation with the Scientific Investigation Department (Carabinieri-RIS) of Rome. The achieved results allow to consider NIR spectroscopy analysis followed by chemometrics as a fast, cost-effective and useful tool for the preliminary determination of new psychoactive substances in forensic science.

Improvement and automation of a real-time PCR assay for vaginal fluids.

The identification of vaginal fluids is crucial in forensic science. Several molecular protocols based on PCR amplification of mfDNA (microflora DNA) specific for vaginal bacteria are now available. Unfortunately mfDNA extraction and PCR reactions require manual optimization of several steps. The aim of present study was the verification of a partial automatization of vaginal fluids identification through two instruments widely diffused in forensic laboratories: EZ1 Advanced robot and Rotor Gene Q 5Plex HRM. Moreover, taking advantage of 5-plex thermocycler technology, the ForFluid kit performances were improved by expanding the mfDNA characterization panel with a new bacterial target for vaginal fluids and with an internal positive control (IPC) to monitor PCR inhibition. Results underlined the feasibility of a semi-automated extraction of mfDNA using a BioRobot and demonstrated the analytical improvements of the kit.

The environmental biological signature: NGS profiling for forensic comparison of soils.

The identification of the source of a specific soil sample is a crucial step in forensic investigations. Rapid advances in next generation sequencing (NGS) technology and the strong reduction of the cost of sequencing have recently opened new perspectives. In the present work a metabarcoding approach has been successfully applied to forensic and environmental soil samples, allowing the accurate and sensitive analysis of microflora (mfDNA), plants, metazoa, and protozoa DNA. The identification of the biological component by DNA metabarcoding is a strong element for the discrimination of samples geologically very similar but coming for distinct environments.

Integrated Ion Beam Analysis (IBA) in Gunshot Residue (GSR) characterisation.

Gunshot Residue (GSR) is residual material from the discharge of a firearm, which frequently provides crucial information in criminal investigations. Changes in ammunition manufacturing are gradually phasing out the heavy metals on which current forensic GSR analysis is based, and the latest Heavy Metal Free (HMF) primers urgently demand new forensic solutions. Proton scanning microbeam Ion Beam Analysis (IBA), in conjunction with the Scanning Electron Microscope equipped with an Energy Dispersive X-ray Spectrometer (SEM-EDS), can be introduced into forensic analysis to solve both new and old problems, with a procedure entirely commensurate with current forensic practice. Six cartridges producing GSR particles known to be interesting in casework by both experience and the literature were selected for this study. A standard procedure to relocate the same particles previously analysed by SEM-EDS, based on both secondary electron (SE) and X-ray imaging was developed and tested. Elemental Particle Induced X-ray Emission (PIXE) mapping of the emitted X-rays allowed relocation in a scan of 10 µm × 10 µm of even a 1 µm GSR particle. The comparison between spectra from the same particle obtained by SEM-EDS and IBA-PIXE showed that the latter is much more sensitive at mid-high energies. Results that are very interesting in a forensic context were obtained with particles from a cartridge containing mercury fulminate in the primer. Particle-induced gamma-ray emission (PIGE) maps of a particles from HMF cartridges allowed identification of Boron and Sodium in particles from hands using the (10)B(p,α1γ)(7)Be, (11)B(p,p1γ)(11)B and (23)Na(p,p1γ)(23)Na reactions, which is extraordinary in a forensic context. The capability for quantitative analysis of elements within individual particles by IBA was also demonstrated, giving the opportunity to begin a new chapter in the research on GSR particles. The integrated procedure that was developed, which makes use of all the IBA signals, has unprecedented characterisation and discrimination power for GSR samples.

Development of a chemiluminescent ELISA and a colloidal gold-based LFIA for TNT detection.

To identify the explosive used in a terrorist attack, or to obtain an early sign of environmental pollution it is important to use simple and rapid assays able to detect analytes at low levels, possibly on-site. This is particularly true for TNT (2,4,6-trinitrotoluene), one of the most employed explosives in the 20th century and at the same time, because of its toxicity, a well known pollutant. In this work we describe the development of an indirect competitive ELISA with chemiluminescent detection (CL-ELISA) and of a lateral-flow immunoassay (LFIA) based on colloidal gold nanoparticle labels. A commercially available monoclonal antibody was used and 13 specially synthesized conjugates were tested. We optimized the assay by determining the optimal concentration of monoclonal antibody and conjugates and the influence of various non-specific factors such as: tolerance to organic solvents at different concentrations, the washing and competitive step time, and the cross-reactivity with related compounds. The sensitivity and reproducibility of the CL-ELISA were good (LOD and IC(50) values in the ng mL(-1) range, and CV value about 7%). It has been applied to real samples of various materials involved in a controlled explosion of an "improvised explosive device". Three extraction procedures were tested on these samples, all employing methanol as the solvent. The lateral flow immunoassay (LFIA), developed by using the same immunoreagents, reached a detection limit of 1 microg mL(-1) when tested on the same samples analysed by CL-ELISA.

Development of chronic pulmonary inflammation in immunized guinea pigs by aerosol challenge with antigen: relationship of immune complex disease and cell-mediated hypersensitivity.

Immunized guinea pigs develop immune complex disease (ICD) in the lungs after a single aerosol challenge with specific antigen. In the current study, immunized guinea pigs developed chronic pulmonary inflammation and cellular immunity (CI) in the lungs when aerosol challenged daily with specific antigen for 2 wk. When immune serum was passively transferred to normal recipients that were then aerosol challenged with specific antigen for 2 wk, chronic pulmonary inflammation and CI did not develop. These results suggest that ICD produced by passive transfer of serum and subsequent aerosol exposure to antigen was inadequate to cause chronic pulmonary inflammation and CI. The development of chronic pulmonary inflammation by aerosol challenge with antigen was suppresed with cobra venom factor. However, because of other studies, we attribute this suppression to the diminution of complement (C) factors in the alternative C pathway that affect macrophage mobility rather than to the depletion of C5a, which is important in the development of ICD.

Enhancement of systemic immune response by immunization into chronically inflamed lungs.

Intrapulmonary instillation of proteins into rabbit lungs with BCG-induced granulomatous inflammation results in greater transport of these molecules into the blood, and the primary route is probably the pulmonary lymphatics. In addition, rabbits with inflamed lungs develop a more potent systemic immune response when exposed to soluble antigens as an aerosol. The current study was done to further study the mechanisms of this phenomenon using the Jerne plaque technique. Intrapulmonary immunization with soluble antigens (solubilized SRBCs and HSA) resulted in a greater PFC response to both antigens when the lungs exhibited BCG-induced granulomatous inflammation. A previous study demonstrated that more antigen administered intratracheally was found in the HLNs when the lungs displayed granulomatous inflammation. However, in the present study, we did not observe an enhanced PFC response in HLN cells when antigens were introduced into inflamed lungs. When rabbits with BCG-inflamed lungs were immunized i.v., they did not develop an enhanced PFC response in the spleen. Immunization into the respiratory tract of normal rabbits with large doses (300 micrograms) of soluble antigens also resulted in a substantial PFC response in the spleen that was quantitatively greater than that induced by the same i.v. dose. These data indicate that (1) administration of antigens into inflamed lung results in an enhanced systemic immune response, (2) although larger quantities of soluble antigens administered by the pulmonary route accumulate in the HLN when lungs are inflamed, cells from this tissue do not exhibit an enhanced PFC response, and (3) large doses of soluble antigens instilled into normal lungs induce a greater systemic immune response that the same doses administered i.v. This study further demonstrates the importance of pulmonary inflammation and the immune response to inhaled antigens and provides insight as to how individuals with chronic inflammatory lung disease can react in an augmented fashion to environmental antigens.

Chromatographic micro-procedure for trace determination of phenobarbital in blood serum.

Volumes of 100 microliters of serum were sufficient for the determination of therapeutic levels of phenobarbital. The isolation procedure was performed using a column method with a hydrophobic adsorbent, graphitized carbon black (Carbopack B). With this method the quantitative (98.1%) recovery of phenobarbital was measured. By suitable choice of experimental conditions, a highly selective purification of the drug can be obtained, thus eliminating various sources of error during quantitation due to the presence in the final samples of endogenous compounds. For the quantitation procedure, another type of graphitized carbon black (Carbopack C) suitably modified was used for gas chromatography. Calibration curves showed no chemisorption effect along the column even on injecting 5 ng of phenobarbital. Some practical aspects of the procedure for improving the reliability of the results are discussed.

Role of angiotensin-converting enzyme in Bacille Calmette-Guérin-induced granulomatous inflammation. Increased angiotensin-converting enzyme levels in lung lavage and suppression of inflammation with captopril.

Lung lavage levels of angiotensin-converting enzyme (ACE)-like activity were increased in C57BL/6 mice with Bacille Calmette-Guérin (BCG)-induced chronic granulomatous pulmonary inflammation and splenomegaly. Contrariwise, ACE activity was not increased in lung lavage fluids of CBA mice that developed only minimal pulmonary inflammation in response to BCG. ACE-like activity correlated with the intensity of inflammation and Captopril, a specific competitive inhibitor of ACE activity, markedly suppressed the induction and maintenance of the BCG-induced inflammatory response in both lungs and spleen. It was necessary, however, to provide sustained treatment with large doses of Captopril in order to reduce the inflammatory response. After a single intraperitoneal injection of Captopril, ACE levels in lung lavage of BCG-injected mice were reduced but returned to preinjection levels or greater within 24 h. The highest dose of Captopril was more effective in reducing the lung fluid level of ACE in BCG-inflamed lungs. This suggests that sustained daily injections of Captopril were necessary to maintain reduced ACE levels. In vitro studies indicated that high concentrations of Captopril did not affect macrophage mobility or chemotactic activity for macrophages. Thus, ACE may act as a molecular mediator of BCG-induced granulomatous inflammation in the lung.