Target and suspect screening of psychoactive substances in seizures and oral fluid exploiting retention time prediction and LC-MS/MS analysis.

BACKGROUND: Novel psychoactive substances (NPS) are a group of substances, mainly of synthetic origin, characterized by toxicological properties extremely dangerous. The main difficulty in recognizing NPS in seizures and biological samples lies in their dynamic nature, related to the continuous synthesis and introduction on the market of new drugs, often with very similar structures to existing ones. The aim of this study was the creation of a robust and versatile method for the analysis of traditional drugs and NPS in different matrices. RESULTS: Both target analysis and suspect screening methodologies were developed. The strategy used for suspect screening allowed to collect data through a scheduled multi reaction monitoring (sMRM) survey which triggered the collection of enhanced product ion (EPI) spectra when a compound met information dependent acquisition (IDA) criteria. The retention time of the different drugs, which was crucial to define the sMRM survey scan parameters, was predicted with a Quantitative Structure Retention (Chromatographic) Relationship (QSRR) model by Multiple Linear Regression. The model was validated through the evaluation of training set predictions, an external validation set and a leave-one out strategy; the results showed that the method fit for its purpose. The target method was validated in oral fluid as a testing matrix, with excellent results in term of recovery, accuracy, precision and matrix effect. Finally, the performances of the suspect method were evaluated by analysing a mixture containing 8 reference standards not included in the initial dataset, as well as seizures and real oral fluid samples. Four NPS were putatively identified in the analysed samples. SIGNIFICANCE: The advantage of the proposed approach is the possibility of quantifying 65 classical drugs of abuse and NPS and, at the same time, detect and putatively identify 146 additional drugs in one single LC-MS/MS run. This is an innovative strategy for multi analyte detection and enables detection of low concentrations of drugs in complex biological matrices such as oral fluid. Considering the highly dynamic drug market, a strength of this strategy is that the analytical method can be kept up to date through the addition of new compounds based on the last drug monitoring bodies alerts without the need of authentic standards.

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.

Efficacy of selective histone deacetylase 6 inhibition in mouse models of Pseudomonas aeruginosa infection: A new glimpse for reducing inflammation and infection in cystic fibrosis.

The latest studies identified the histone deacetylase (HDAC) class of enzymes as strategic components of the complex molecular machinery underlying inflammation in cystic fibrosis (CF). Compelling new support has been provided for HDAC6 isoform as a key player in the generation of the dysregulated proinflammatory phenotype in CF, as well as in the immune response to the persistent bacterial infection accompanying CF patients. We herein provide in vivo proof-of-concept (PoC) of the efficacy of selective HDAC6 inhibition in contrasting the pro-inflammatory phenotype in a mouse model of chronic P. aeruginosa respiratory infection. Upon careful selection and in-house re-profiling (in vitro and cell-based assessment of acetylated tubulin level through Western blot analysis) of three potent and selective HDAC6 inhibitors as putative candidates for the PoC, we engaged the best performing compound 2 for pre-clinical studies. Compound 2 demonstrated no toxicity and robust anti-inflammatory profile in a mouse model of chronic P. aeruginosa respiratory infection upon repeated aerosol administration. A significant reduction of leukocyte recruitment in the airways, in particular neutrophils, was observed in compound 2-treated mice in comparison with the vehicle; moreover, quantitative immunoassays confirmed a significant reduction of chemokines and cytokines in lung homogenate. This effect was also associated with a modest reduced bacterial load after compound 2-treatment in mice compared to the vehicle. Our study is of particular significance since it demonstrates for the first time the utility of selective drug-like HDAC6 inhibitors in a relevant in vivo model of chronic P. aeruginosa infection, thus supporting their potential application for reverting CF phenotype.

Beneficial Contribution to Glucose Homeostasis by an Agro-Food Waste Product Rich in Abscisic Acid: Results from a Randomized Controlled Trial.

The control of glucose homeostasis represents the primary goal for the prevention and management of diabetes and prediabetes. In recent decades, the hypoglycemic hormone abscisic acid (ABA) has attracted considerable interest in the scientific literature. In this regard, the high ABA concentration in immature fruits led us to consider these food matrices as candidates for diabetes control. Therefore, the beneficial efficacy of a nutraceutical formulation based on thinned nectarines (TNs) rich in ABA was tested through a three-month, three-arm, parallel-group, randomized controlled trial (RCT) conducted on sixty-one patients with type 2 diabetes (T2D). After 3 months, both the treatments with low doses of TN (500 mg 3 times/day) and high doses of TN (750 mg 3 times/day) showed a significant reduction in glycemic parameters compared to baseline. Treatment with low doses of TN showed a greater insulin-sparing effect (fasting plasma insulin, FPI: −29.2%, p < 0.05 vs. baseline) compared to the high-dose group (FPI: −16.5%, p < 0.05 vs. baseline). Moreover, a significant correlation between glycemia and ABA plasmatic levels was observed for both intervention groups at baseline and after 3 months. Overall, our data reasonably support TN as a promising and innovative nutraceutical product able to contribute to the management of glucose homeostasis.