Quantitative analysis of fentanyl, several analogues and metabolites in urine by parallel artificial liquid membrane extraction and liquid chromatography tandem mass spectrometry analysis

The rapid introduction of new psychoactive substances (NPS) has definitively changed the drug market. Among the several NPS that were identified in the last decades, fentanyl and its analogues deserve special attention. These are synthetic opioids with high potency and are associated with increasing number of deaths; for this reason, forensic toxicologists are paying close attention to these analytes and sensitive analytical methods for their detection in biological samples of drug users are needed. The aim of this study was the development of a LC–MS/MS method for the determination of fentanyl, 23 analogues and metabolites in urine by exploiting parallel artificial liquid membrane extraction (PALME). This technique was shown to be particularly suitable for fentanyl extraction and allowed to obtain a high enrichment factor by using a few microliters of organic solvent (1-octanol) immobilized into a polyvinylidene fluoride (PVDF) membrane. The extraction was carried out on a 96 well plate providing high laboratory throughput. The applied strategy allowed to measure concentrations ranging from 0.1 ng mL − 1 for fentanyl and most analogues to 5 ng mL − 1 for metabolites, by using an entry level mass spectrometer. Because of the different concentration levels generally found in real samples, linearity was studied in different ranges i.e. LOQ to 50 ng mL − 1 for parent drugs and LOQ to 200 ng mL − 1 for metabolites. All the validation parameters were found within the imposed limits, and notably matrix effect was not significant for all the analytes, showing the selectivity achieved by PALME extraction

Inside the history of Italian coloring industries. An investigation of ACNA dyes through a novel analytical protocol for synthetic dye extraction and characterization

The introduction of synthetic dyes completely changed the industrial production and use of colorants for art materials. From the synthesis of the first synthetic dye, mauveine, in 1856 until today, artists have enjoyed a wider range of colors and selection of chemical properties than was ever available before. However, the introduction of synthetic dyes introduced a wider variety and increased the complexity of the chemical structures of marketed dyes. This work looks towards the analysis of synthetically dyed objects in heritage collections, applying an extraction protocol based on the use of ammonia, which is considered favorable for natural anthraquinone dyes but has never before been applied to acid synthetic dyes. This work also presents an innovative cleanup step based on the use of an ion pair dispersive liquid–liquid microextraction for the purification and preconcentration of historical synthetic dyes before analysis. This approach was adapted from food science analysis and is applied to synthetic dyes in heritage science for the first time in this paper. The results showed adequate recovery of analytes and allowed for the ammonia-based extraction method to be applied successfully to 15 samples of suspected azo dyes from the Azienda Coloranti Nazionali e Affini (ACNA) synthetic dye collection, identified through untargeted HPLC-HRMS analyses

New advances in dye analyses. In situ gel-supported liquid extraction from paint layers and textiles for SERS and HPLC-MS/MS Identification

To date, it is still not possible to obtain exhaustive information about organic materials in cultural heritage without sampling. Nonetheless, when studying unique objects with invaluable artistic or historical significance, preserving their integrity is a priority. In particular, organic dye identification is of significant interest for history and conservation research, but it is still hindered by analytes’ low concentration and poor fastness. In this work, a minimally invasive approach for dye identification is presented. The procedure is designed to accompany noninvasive analyses of inorganic substances for comprehensive studies of complex cultural heritage matrices, in compliance with their soundness. Liquid extraction of madder, turmeric, and indigo dyes was performed directly from paint layers and textiles. The extraction was supported by hydrogels, which themselves can undergo multitechnique analyses in the place of samples. After extraction, Ag colloid pastes were applied on the gels for SERS analyses, allowing for the identification of the three dyes. For the HPLC-MS/MS analyses, re-extraction of the dyes was followed by a clean-up step that was successfully applied on madder and turmeric. The colour change perceptivity after extraction was measured with colorimetry. The results showed ΔE values mostly below the upper limit of rigorous colour change, confirming the gentleness of the procedure

Untargeted Metabolic Profiling of 4-Fluoro-Furanylfentanyl and Isobutyrylfentanyl in Mouse Hepatocytes and Urine by Means of LC-HRMS

The diffusion of new psychoactive substances (NPS) is highly dynamic and the available substances change over time, resulting in forensic laboratories becoming highly engaged in NPS control. In order to manage NPS diffusion, efficient and innovative legal responses have been provided by several nations. Metabolic profiling is also part of the analytical fight against NPS, since it allows to identify the biomarkers of drug intake which are needed for the development of suitable analytical methods in biological samples. We have recently reported the characterization of two new analogs of fentanyl, i.e., 4-fluoro-furanylfentanyl (4F-FUF) and isobutyrylfentanyl (iBF), which were found for the first time in Italy in 2019; 4F-FUF was identified for the first time in Europe and was notified to the European Early Warning System. The goal of this study was the characterization of the main metabolites of both drugs by in vitro and in vivo experiments. To this end, incubation with mouse hepatocytes and intraperitoneal administration to mice were carried out. Samples were analyzed by means of liquid chromatography-high resolution mass spectrometry (LC–HRMS), followed by untargeted data evaluation using Compound Discoverer software with a specific workflow, designed for the identification of the whole metabolic pattern, including unexpected metabolites. Twenty metabolites were putatively annotated for 4F-FUF, with the dihydrodiol derivative appearing as the most abundant, whereas 22 metabolites were found for iBF, which was mainly excreted as nor-isobutyrylfentanyl. N-dealkylation of 4F-FUF dihydrodiol and oxidation to carbonyl metabolites for iBF were also major biotransformations. Despite some differences, in general there was a good agreement between in vitro and in vivo samples

Unearthed opium. Development of a UHPLC-MS/MS method for the determination of Papaver somniferum alkaloids in Daunian vessels

Introduction: The analysis of organic residue in ancient vessels to investigate early-age civilization habits is an important archeological application that needs advanced analytical methods. However, these procedures should meet inherent requisites such as low sampling invasiveness and high sensitivity for trace analysis. This study deals with the development of advanced analytical methods for the detection of opium alkaloids in ceramic vessels and its first application to the study of Daunian pots dating back to the VIII-IV sec BC.Methods: All the stages of the analytical procedure, from sampling to analysis, were carefully optimized. Concerning sampling, the traditional scraping approach was compared with a swabbing strategy which permitted minimizing sample encroachment. Extraction was based on pressurized liquid extraction or ultrasound-assisted liquid extraction, followed by dispersive liquid-liquid microextraction, which allowed concentration enrichment. On the other hand, a UHPLC-MS/MS method was specifically developed and validated to obtain reliable data. Some Daunian pots, belonging to the Ceci-Macrini private archeological collection, were selected for sample withdrawal as their iconography could suggest opium usage.Results: Several of the analyzed samples resulted positive to thebaine and less frequently to morphine and codeine; furthermore, 70% of the analyzed items tested positive for at least one opium alkaloid. Positive findings were common to all the samples collected in the pots, suggesting that scraping and swabbing provided comparable results and validating this unusual sampling strategy. All samples were additionally analyzed by UHPLC-HRMS to further improve the confidence level of the identified compounds. The obtained results shed new light on the hypothesis of opium usage by the ancient Daunian civilization. Furthermore, this study provided suitable analytical tools for further investigations on the same topic, with a good level of confidence in the quality of the results

New synthetic opioids: development of analytical methods for their characterization and determination by means of (U)HPLC- HRMS/MS

In recent years, the synthesis and introduction into the illicit market of New Psychoactive Substances (NPS) has reached alarming levels. More than 790 compounds have been identified by the European Monitoring Center for Drugs and Drug Addiction. Among the newest NPS, synthetic opioids deserve special attention, in particular fentanyls, which covered more than 70% of the opioid’s world demand. In this context it is essential to have the right tools to identify these recent NPS and verify their consumption. The present project involved the characterization of new fentanyls, the identification of their main metabolites and the development of innovative, fast and simple analytical methods, for the determination of these compounds in different biological matrices. Results presented in the experimental part illustrates the multi-pronged strategy that was studied in this 3-years project. On the one hand miniaturized preparation techniques, such as micro extraction by packed sorbent (MEPS), parallel artificial liquid membrane extraction (PALME), were optimized for the analyses of fentanyl, fentalogs and their metabolites, conducted in HPLC- (HR)MS/MS. All methods involved the use of a small amount of biological sample (100 μL) On the other hand, advanced data mining tools for untargeted analysis involving molecular networking were investigated and allowed to connect unknown compounds to “standard networks,” simplifying the annotation of new drugs. Different complementary analytical techniques, such as IR, Raman, GC-MS, LC-HRMS/MS and NMR were then applied to characterize the unknown substances and for the first time isobutyrylfentanyl (iBF) and 4-flouro- furanylfentanyl (4F-FuF) were characterized. Metabolic profiles of iBF and 4F-FuF were investigated by means of in silico, in vitro and in vivo studies, and their major metabolites were identified for the first time. For the first compound the N-dealkylated metabolite was identified as the main biomarker, while for 4F-FUF, the main metabolite was the dihydrodiol derivative, which was further N-dealkylated. And then, thanks to the pharmaco-toxicological studies, it was possible to study the distribution of the two drugs in the different tissues. The project was carried out in collaboration with the Scientific Investigations Department of the Arma dei Carabinieri of Rome. Unpayable support was also provided by Professor Marti, head of the toxicology unit of the University of Ferrara. Thanks to this work, the knowledge of this particular class of NPS and the associated metabolic processes has been expanded, providing a fundamental contribution to forensic science as well as pharmaco-toxicology. Furthermore, it was possible to make an effective contribution to the estimate of the real propagation of NPS in the European illegal market

Accelerated Extraction and Analysis of Ethyl Glucuronide in Hair by Means of Pressurized Liquid Extraction Followed by Liquid Chromatography–Tandem Mass Spectrometry Determination

The measurement of ethyl glucuronide (EtG) in hair is an established practice to evaluate alcohol consumption habits of the donors, nevertheless analytical variability has shown to be an important factor to be considered: measured EtG values can vary significantly as a consequence of analyte wash-out during decontamination, pulverization of samples, extraction solvent and incubation temperature. In the present study we described a new method for automated hair decontamination and EtG extraction from the inner core of the hair by using pressurized liquid extraction (PLE), followed by solid phase extraction (SPE) clean-up; validation was performed according to SWGTOX guidelines. The extraction efficiency of the new method was evaluated by comparing the results with those obtained by a validated and ISO/IEC 17025:2005 accredited method; an average positive difference of +32% was observed when the extraction was performed by PLE. The effect of hair pulverization was also studied and a good correlation between cut and milled hair was observed, implying that PLE allowed a highly efficient extraction of EtG from the inner keratin core of the hair, no matter if it has been cut or pulverized. Finally, to verify the results, paired aliquots of 27 real hair samples were analyzed with both PLE and a protocol optimized by design-of-experiment strategies planned to maximize the extraction yield; in this case a comparable efficiency was observed, suggesting that exhaustive EtG extraction was obtained with both approaches. This finding opens new perspectives in the eligible protocols devoted to hair EtG analysis, in terms of speed, automation, and reproducibility