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Set up of a simple, fast and cheap liquid chromatographic method to separate four macrolides and to quantify their concentrations. It was optimised evaluating mobile phase composition, column temperature and pH to provide the best resolution of these analytes. Preliminary assays are promising and the method is under validation for application to real food samples.

Important components of the palm fruit such as oils, fatty acids, semi-solid fats, vitamin E, tocopherols, carotenoids, phytosterols and flavonoids, are here identified and quantified for their importance in the nutraceutical and food industry.

Aim of this work is to develop an innovative, feasible but reliable qualitative and quantitative analysis of Ketamine and its active metabolite Norketamine in saliva samples, optimizing fast and effective pretreatment procedures and using HPLC coupled to different detection means such as spectrofluorimetry (F) and tandem mass spectrometry (MS/MS). Separation is achieved by means of RP C18 stationary phase and a mobile phase consisting in a mixture of acetonitrile and acidic aqueous buffer. The pretreatment of saliva samples is obtained by a miniaturized system of Micro Extraction by Packed Sorbent (MEPS). The preliminary results are promising and assays are in progress in order to fully validate the method.

Valorization of wild plants to obtain botanical ingredients could be a strategy for sustainable production of cosmetics. This study aimed to select the rosehip extract containing the greatest amounts of bioactive compounds and to encapsulate it in vesicular systems capable of protecting their own antioxidant activity. Chemical analysis of Rosa canina L. extracts was performed by LC-DAD-MS/MS and 1H-NMR and vitamins, phenolic compounds, sugars, and organic acids were detected as the main compounds of the extracts. Liposomes, prepared by the film hydration method, together with hyalurosomes and ethosomes, obtained by the ethanol injection method, were characterized in terms of vesicle size, polydispersity index, entrapment efficiency, zeta potential, in vitro release and biocompatibility on WS1 fibroblasts. Among all types of vesicular systems, ethosomes proved to be the most promising nanocarriers showing nanometric size (196 ± 1 nm), narrow polydispersity (0.20 ± 0.02), good entrapment efficiency (92.30 ± 0.02%), and negative zeta potential (−37.36 ± 0.55 mV). Moreover, ethosomes showed good stability over time, a slow release of polyphenols compared with free extract, and they were not cytotoxic. In conclusion, ethosomes could be innovative carriers for the encapsulation of rosehip extract.

This review summarizes the current knowledge on essential vitamins B1, B2, B3, and B5. These B-complex vitamins must be taken from diet, with the exception of vitamin B3, that can also be synthetized from amino acid tryptophan. All of these vitamins are water soluble, which determines their main properties, namely: they are partly lost when food is washed or boiled since they migrate to the water; the requirement of membrane transporters for their permeation into the cells; and their safety since any excess is rapidly eliminated via the kidney. The therapeutic use of B-complex vitamins is mostly limited to hypovitaminoses or similar conditions, but, as they are generally very safe, they have also been examined in other pathological conditions. Nicotinic acid, a form of vitamin B3, is the only exception because it is a known hypolipidemic agent in gram doses. The article also sums up: (i) the current methods for detection of the vitamins of the B-complex in biological fluids; (ii) the food and other sources of these vitamins including the effect of common processing and storage methods on their content; and (iii) their physiological function.

Despite the long and sometimes enthusing history of pharmacological therapy in psychiatry, the hard truth is that unfortunately a quite large percentage of patients is still not responding, or poorly responding, to treatment, leading to many life years lost to disability, many lost lives, and an immeasurable amount of suffering from patients, relatives, friends, and caregivers alike. Thus, any scientific advance and any practice that could lead to even a slight increase in psychiatric therapy effectiveness would also bring with them enormous benefits for both citizens and healthcare institutions. It is a strong conviction, after many years of practice and study, that therapeutic drug monitoring (TDM) is one of these practices, one that is continually advancing and progressing both from the analytical and clinical points of view, toward the final goal of better, personalized, precision medicine.

Vitamin C (L-ascorbic acid) has been known as an antioxidant for most people. However, its physiological role is much larger and encompasses very different processes ranging from facilitation of iron absorption through involvement in hormones and carnitine synthesis for important roles in epigenetic processes. Contrarily, high doses act as a pro-oxidant than an anti-oxidant. This may also be the reason why plasma levels are meticulously regulated on the level of absorption and excretion in the kidney. Interestingly, most cells contain vitamin C in millimolar concentrations, which is much higher than its plasma concentrations, and compared to other vitamins. The role of vitamin C is well demonstrated by miscellaneous symptoms of its absence—scurvy. The only clinically well-documented indication for vitamin C is scurvy. The effects of vitamin C administration on cancer, cardiovascular diseases, and infections are rather minor or even debatable in the general population. Vitamin C is relatively safe, but caution should be given to the administration of high doses, which can cause overt side effects in some susceptible patients (e.g., oxalate renal stones). Lastly, analytical methods for its determination with advantages and pitfalls are also discussed in this review.

A glassy carbon electrode chemically modified with a carbon black coating is proposed here for the rapid and portable determination of cannabidiol (CBD) in a commercial Cannabis seed oil and in fibre-type Cannabis sativa L. leaves. The mechanism of CBD oxidation was studied in relation to simpler phenyl derivatives bearing the same electroactive group, namely resorcinol and 2-methylresorcinol. These molecules also allowed us to determine the best conditions for the electrochemical detection of CBD, as to the pH value and to the best solvent mixture to use. Carbon black was chosen among nanostructured carbon-based materials owing to its outstanding features as an electrode modifier for analyte detection. The performance of the modified electrode was determined by flow injection analyses of standard solutions of CBD, obtaining a linear correlation between the oxidation current and the analyte concentration; the sensor response is characterised by suitable repeatability and reproducibility. The analysis of commercial products by the standard addition method allowed us to ascertain the accuracy of the sensor for the detection of CBD in real samples.

Clenbuterol is a chiral, selective β2-adrenergic agonist. It is administered as a racemic mixture for therapeutic purposes (as a bronchodilator or prospective neuroprotective agent), but also for non-therapeutic uses (athletic performance enhancement, cattle growth promotion). Aim of the present study is to develop an original, enantioselective workflow for the analysis of clenbuterol enantiomers in urine microsamples. An innovative miniaturised sampling procedure by volumetric absorptive microsampling (VAMS) and a microsample pretreatment strategy based on stop-and-go extraction (StAGE) tips were developed and coupled to an original, chiral analytical method, exploiting liquid chromatography with triple quadrupole detection (LC-MS/MS). The method was validated, with satisfactory results: good linearity (r2 ≥ 0.9995) and LOQ values (0.3 ng/mL) were found over suitable concentration ranges. Extraction yield (>87 %), precision (RSD < 4.3 %) and matrix effect (85–90 %) were all within acceptable levels of confidence. After validation, the method was applied to the determination of clenbuterol in dried urine sampled by VAMS from patients taking the drug for therapeutic reasons. Analyte content ranged from 0.8 to 2.5 ng/mL per single enantiomer, with substantial retention of the original drug racemic composition. The VAMS-StAGE-LC-MS/MS workflow seems to be suitable for future application to anti-doping testing of clenbuterol in urine.

Cannabidiol (CBD) is a bioactive terpenophenolic compound isolated from Cannabis sativa L. It is known to possess several properties of pharmaceutical interest, such as antioxidant, anti-inflammatory, anti-microbial, neuroprotective and anti-convulsant, being it active as a multi-target compound. From a therapeutic point of view, CBD is most commonly used for seizure disorder in children. CBD is present in both medical and fiber-type C. sativa plants, but, unlike Δ9-tetrahydrocannabinol (THC), it is a non-psychoactive compound. Non-psychoactive or fiber-type C. sativa (also known as hemp) differs from the medical one, since it contains only low levels of THC and high levels of CBD and related non-psychoactive cannabinoids. In addition to medical Cannabis, which is used for many different therapeutic purposes, a great expansion of the market of hemp plant material and related products has been observed in recent years, due to its usage in many fields, including food, cosmetics and electronic cigarettes liquids (commonly known as e-liquids). In this view, this work is focused on recent advances on sample preparation strategies and analytical methods for the chemical analysis of CBD and related compounds in both C. sativa plant material, its derived products and biological samples. Since sample preparation is considered to be a crucial step in the development of reliable analytical methods for the determination of natural compounds in complex matrices, different extraction methods are discussed. As regards the analysis of CBD and related compounds, the application of both separation and non-separation methods is discussed in detail. The advantages, disadvantages and applicability of the different methodologies currently available are evaluated. The scientific interest in the development of portable devices for the reliable analysis of CBD in vegetable and biological samples is also highlighted.

Volumetric absorptive microsampling (VAMS) and dried urine spot (DUS) strategies were applied for the collection of dried microsamples for anti-doping testing of low-stability peptide hormones and growth factors prohibited by the World Anti-Doping Agency (WADA). Drying, storage and transport conditions, as well as pretreatment steps, were optimised before liquid chromatography - tandem mass spectrometry (LC–MS/MS) analysis. The analytical method has been fully validated in terms of sensitivity (limits of quantitation 0.3−10 ng/mL), precision (RSD% < 6.6 %) and extraction yields (78–91 %). Dried microsample stability studies (90 days) have been performed and compared to fluid urine stability. Significantly higher losses have been observed in fluid urine stored at −20 °C (up to 55 %) and −80 °C (up to 29 %) than in dried urine microsamples stored at room temperature (< 19 %). The final microsampling and analysis protocols allow the collection of urine microvolumes, unlikely to be tampered, stably storable and shippable with no particular precautions for possible anti-doping testing of prohibited peptides and hormones.

Amikacin (Amk) analysis and quantitation, for pharmacokinetics studies and other types of investigations, is conventionally performed after extraction from plasma. No report exists so far regarding drug extraction from whole blood (WB). This can represent an issue since quantification in plasma does not account for drug partitioning to the blood cell compartment, significantly underrating the drug fraction reaching the blood circulation. In the present work, the optimization of an extraction method of Amk from murine WB has been described. The extraction yield was measured by RP-HPLC-UV after derivatization with 1-fluoro-2,4-dinitrobenzene, which produced an appreciably stable derivative with a favorable UV/vis absorption. Several extraction conditions were tested: spiked Amk disulfate solution/acetonitrile/WB ratio; presence of organic acids and/or ammonium hydroxide and/or ammonium acetate in the extraction mixture; re-dissolution of the supernatant in water after a drying process under vacuum; treatment of the supernatant with a solution of inorganic salts. The use of 5% (by volume) of ammonium hydroxide in a hydro-organic solution with acetonitrile, allowed the almost quantitative (95%) extraction of the drug from WB.

In the present, proof-of-concept paper, we explore the potential of one common solid support for blood microsampling (dried blood spot, DBS) and a device (volumetric absorptive microsampling, VAMS) developed for the untargeted lipidomic profiling of human whole blood, performed by high-resolution LC-MS/MS. Dried blood microsamples obtained by means of DBS and VAMS were extracted with different solvent compositions and compared with fluid blood to evaluate their efficiency in profiling the lipid chemical space in the most broad way. Although more effort is needed to better characterize this approach, our results indicate that VAMS is a viable option for untargeted studies and its use will bring all the corresponding known advantages in the field of lipidomics, such as haematocrit independence.

Fentanyl analogues used in therapy and a range of highly potent non-pharmaceutical fentanyl derivatives are subject to international control, as the latter are increasingly being synthesized illicitly and sold as ‘synthetic heroin’, or mixed with heroin. A significant number of hospitalizations and deaths have been reported in the EU and USA following the use of illicitly synthesized fentanyl derivatives. It has been unequivocally demonstrated that the enantiomers of fentanyl derivatives exhibit different pharmaco-toxicological profiles, which makes crucial to avail of suitable analytical methods enabling investigations at a “stereochemical level”. Chromatographic methods useful to discriminate the enantioseparation of fentanyls and their derivatives are still missing in the literature. This is the first study in which the enantioseparation of four fentanyl derivatives, that is, (±)-trans-3-methyl norfentanyl, (±)-cis-3-methyl norfentanyl, β-hydroxyfentanyl, and β-hydroxythiofentanyl, has been obtained under polar-ionic conditions. Indeed, the use of ACN-based mobile phases with minor amounts of either 2-propanol or ethanol (plus diethylamine and formic acid as ionic additives) allowed obtaining enantioseparation and enantioresolution factors up to 1.83 and 7.02, respectively. For the study, the two chiral stationary phases cellulose tris(3-chloro-4-methylphenylcarbamate) and cellulose tris(4-chloro-3-methylphenylcarbamate) were used, displaying a remarkably different performance towards the enantioseparation of (±)-cis-3-methyl norfentanyl. Chiral LC analyses with a high-resolution mass spectrometry detector were also carried out in order to confirm the obtained data and demonstrate the suitability and compatibility of the optimized mobile phases with mass spectrometric systems.

Cannabis sativa L. is a plant known all over the world, due to its history, bioactivity and also social impact. It is chemically complex with an astonishing ability in the biosynthesis of many secondary metabolites belonging to different chemical classes. Among them, cannabinoids are the most investigated ones, given their pharmacological relevance. In order to monitor the composition of the plant material and ensure the efficacy and safety of its derived products, extraction and analysis of cannabinoids play a crucial role. In this context, in addition to a conventional separation method based on HPLC with UV/DAD detection, a new strategy based on a non-separation procedure, such as 13C-qNMR, may offer several advantages, such as reduced solvent consumption and simultaneous acquisition of the quali/quantitative data related to many analytes. In the light of all the above, the aim of this work is to compare the efficiency of the above-mentioned analytical techniques for the study of the main cannabinoids in different samples of cannabis inflorescences, belonging to fibre-type, recreational and medical varieties. The 13C-qNMR method here proposed for the first time for the quantification of both psychoactive and non-psychoactive cannabinoids in different cannabis varieties provided reliable results in comparison to the more common and consolidated HPLC technique.

Single enantiomers of mandelic acid (1), 3-phenyllactic acid (2), and 3-(4-hydroxyphenyl)lactic acid (3) are the subject of many fields of investigation, spanning from the pharmaceutical synthesis to that of biocompatible and biodegradable polymers, while passing from the interest towards their antimicrobial activity to their role as biomarkers of particular pathological conditions or occupational exposures to specific xenobiotics. All above mentioned issues justify the need for accurate analytical methods enabling the correct determination of the individual enantiomers. So far, all the developed liquid chromatography (LC) methods were not or hardly compatible with mass spectrometry (MS) detection. In this paper, a commercially available Cinchona-alkaloid derivative zwitterionic chiral stationary phase [that is, the CHIRALPAK® ZWIX(−)] was successfully used to optimize the enantioresolution of compounds 1–3 under polar-ionic (PI) conditions with a mobile phase consisting of an acetonitrile/methanol 95/5 (v/v) mixture with 80 mM formic acid. With the optimized conditions, enantioseparation and enantioresolution values up to 1.46 and 4.41, respectively, were obtained. In order to assess the applicability of the optimized enantioselective chromatography conditions in real-life scenarios and on MS-based systems, a proof-of-concept application was efficiently carried out by analysing dry urine spot samples spiked with 1 by means of a LC-MS system. The (S)

Testing and monitoring anabolic androgenic steroids in biological fluids is a key activity in anti-doping practices. In this study, a novel approach is proposed, based on dried urine microsampling through two different workflows: dried urine spots (DUS) and volumetric absorptive microsampling (VAMS). Both techniques can overcome some common drawbacks of urine sampling, such as analyte instability and storage and transportation problems. Using an original, validated liquid chromatography&ndash, tandem mass spectrometry (LC-MS/MS) method, exogenous and endogenous unconjugated steroids were analysed. Despite the limitations of microsampling volume, good sensitivity was obtained (limit of quantitation &le, 1.5 ng/mL for all analytes), with satisfactory precision (relative standard deviation &lt, 7.6%) and absolute recovery (&gt, 70.3%). Both microsampling platforms provide reliable results, in good agreement with those obtained from urine.

A new Thymus vulgaris L. solid essential oil (SEO) formulation composed of liquid EO linked to solid excipients has been chemically analysed and evaluated for its intestinal spasmolytic and antispastic effects in ex vivo ileum and colon of guinea pig and compared with liquid EO and excipients. Liquid EO and solid linked EO were analysed by original capillary electrochromatography coupled to diode array detection (CEC-DAD) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) methodologies. The main bioactive constituents are thymol and carvacrol, with minor constituents for a total of 12 selected analysed compounds. Liquid EO was the most effective in decreasing basal contractility in ileum and colon, excipients addiction permitted normal contractility pattern in solid linked EO SEO. In ileum and colon, the Thymus vulgaris L. solid formulation exerted the relaxant activity on K+-depolarized intestinal smooth muscle as well as liquid EO. The solid essential oil exhibits antimicrobial activity against different strains (Staphylococcus aureus, Streptococcus pyogenes, Pseudomonas aeruginosa, Escherichia coli, Salmonella Thyphimurium, Candida albicans) similarly to liquid oil, with activity against pathogen, but not commensal strains (Bifidobacterium Breve, Lactobacillus Fermentum) in intestinal homeostasis. Therefore, Thymus vulgaris L. solid essential oil formulation can be proposed as a possible spasmolytic and antispastic tool in medicine.

Graphical abstract, Highlights • SARS-CoV-2 emergency sparks the need for diagnostic and therapeutic actions. • Microsampling is emerging in as an attractive alternative to traditional sampling. • Advantages and challenges of the main microsampling techniques are reported. • Available microsampling applications of interest for SARS-CoV-2 are described. • Most useful information for researchers and clinicians are gathered and provided., The multiple pathological effects of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, and its total novelty, mean that currently a lot of diagnostic and therapeutic tools, established and tentative alike, are needed to treat patients in a timely, effective way. In order to make these tools more reliable, faster and more feasible, biological fluid microsampling techniques could provide many advantages. In this review, the most important microsampling techniques are considered (dried matrix spots, volumetric absorptive microsampling, microfluidics and capillary microsampling, solid phase microextraction) and their respective advantages and disadvantages laid out. Moreover, currently available microsampling applications of interest for SARS-CoV-2 therapy are described, in order to make them as much widely known as possible, hopefully providing useful information to researchers and clinicians alike.

Lactobacillus spp. dominating the vaginal microbiota of healthy women contribute to the prevention of urogenital and sexually transmitted infections. Their protective role in the vagina can be mediated by Lactobacillus cells themselves, metabolites or bacterial components, able to interfere with pathogen adhesion and infectivity. Vulvovaginal candidiasis (VVC) is a common genital infection, caused by the overgrowth of opportunistic Candida spp. including C. albicans, C. glabrata, C. krusei and C. tropicalis. Azole antifungal drugs are not always efcient in resolv ing VVC and preventing recurrent infections, thus alternative anti-Candida agents based on vaginal probiotics have gained more importance. The present work aims to chemically characterize the biosurfactant (BS) isolated from a vaginal Lactobacillus crispatus strain, L. crispatus BC1, and to investigate its safety and antiadhesive/antimicrobial activ ity against Candida spp., employing in vitro and in vivo assays. Results: BS isolated from vaginal L. crispatus BC1 was characterised as non-homogeneous lipopeptide molecules with a critical micellar concentration value of 2 mg/mL, and good emulsifcation and mucoadhesive properties. At 1.25 mg/mL, the BS was not cytotoxic and reduced Candida strains? ability to adhere to human cervical epithelial cells, mainly by exclusion mechanism. Moreover, intravaginal (i.va.) inoculation of BS in a murine experimental model was safe and did not perturb vaginal cytology, histology and cultivable vaginal microbiota. In the case of i.va. challenge of mice with C. albicans, BS was able to reduce leukocyte infux. Conclusions: These results indicate that BS from vaginal L. crispatus BC1 is able to interfere with Candida adhesion in vitro and in vivo, and suggest its potential as a preventive agent to reduce mucosal damage occasioned by Candida during VVC. Fil: de Gregorio, Priscilla Romina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Parolin, Carola. Universidad de Bologna; Italia Fil: Abruzzo, Angela. Universidad de Bologna; Italia Fil: Luppi, Barbara. Universidad de Bologna; Italia Fil: Protti, Michele. Universidad de Bologna; Italia Fil: Mercolini, Laura. Universidad de Bologna; Italia Fil: Silva, Jessica Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Giordani, Barbara. Universidad de Bologna; Italia Fil: Marangoni, Antonella. Universidad de Bologna; Italia Fil: Nader, Maria Elena Fatima. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Vitali, Beatrice. Universidad de Bologna; Italia

Multiple sclerosis (MS) is a complex inflammatory, degenerative, and demyelinating disease of the central nervous system. Although treatments exist, MS cannot be cured by available drugs, which primarily target neuroinflammation. Thus, it is feasible that a well concerted polypharmacological approach able to act at multiple points within the intricate network of inflammation, neurodegeneration, and demyelination/remyelination pathways would succeed where other drugs have failed. Starting from reported beneficial effects of α-linolenic acid (ALA) and valproic acid (VPA) in MS, and by applying a rational strategy, we developed a small set of codrugs obtained by conjugating VPA and ALA through proper linkers. A cellular profiling identified 1 as a polypharmacological tool able not only to modulate microglia polarization, but also to counteract neurodegeneration and demyelination and induce oligodendrocyte precursor cell differentiation, by acting on multiple biochemical and epigenetic pathways.

In this paper, two new reversed-phase (RP) HPLC-UV methods enabling the quantitative achiral and chiral analysis of ruxolitinib in commercial tablets (containing 20 mg of active pharmaceutical ingredient, API) and not commercially available galenic capsules (with 5 mg of API) are described. For the achiral method based on the use of a water/acetonitrile [70:30, v/v; with 0.1% (v) formic acid] eluent, a “research validation” study was performed mostly following the “International Council for Harmonization” guidelines. All the system suitability parameters were within the acceptance criteria: tailing factor, between 1.7 and 2.0; retention factor, 2.2; number of theoretical plates, >9000. The linearity curve showed R2 = 0.99 (Rxv2 = 0.99), while trueness (expressed as recovery) was between 96.3 and 106.3%. Coefficient of variations (CVs) (repeatability: CVw and intermediate precision: CVIP) did not exceed 1.3% and 2.9%, respectively. Moreover, the use of the enantiomeric Whelk-O1 chiral stationary phases operated under similar RP eluent conditions as for the achiral analyses, and the “inverted chirality columns approach (ICCA)” allowed us to establish that the enantiomeric purity of ruxolitinib is retained upon reformulation from tablets to capsules. The two developed methods can allow accurate determinations of ruxolitinib in drug formulations for medical use.

The new ultra-high performance liquid chromatography method with tandem mass spectrometry detection (UHPLC-MS/MS) has been optimized to allow fast, selective, and high-throughput analysis of two Candida albicans quorum sensing molecules (QSM), farnesol and tyrosol. The problem of the presence of the interference in the samples and system was successfully solved by careful optimization of chromatographic conditions. Charged hybrid stationary phase modified with pentafluorophenyl group and optimized gradient elution provided adequate separation selectivity and peak shapes. The impurity was identified as dibutyl phthalate and had the same m/z ions as farnesol leading to an important interference on selected reaction monitoring channel. Two different types of biological matrices originating from vaginal fluid, supernatant and sediment, were analysed. Micro-solid phase extraction in pipette tips was optimized for the selective isolation of QSM from the supernatant. The insufficient retention of farnesol on the extraction sorbent was improved when 1% of organic solvent was added prior to extraction, while the retention of tyrosol was only possible when using combined C8 and polymer sorbent type. Strong retention of farnesol had to be solved by increasing elution solvent strength and volume up to 600μL. However, this approach did not allow the pretreatment of sediment samples due to the sorbent clogging. Therefore, our previously developed protein precipitation method was modified and validated to analyse the sediments. New developed UHPLC-MS/MS method provided suitable accuracy and precision for the determination of QSM in vaginal fluid while using only 50μL sample volume and two different sample preparation methods.

The aim of this work is to provide a critical review of plant furanocoumarins from different points of view, including their chemistry and biosynthetic pathways to their extraction, analysis, and synthesis, to the main biological activities found for these active compounds, in order to highlight their potential within pharmaceutical science. The limits and the possible improvements needed for research involving these molecules are also highlighted and discussed.

The argan tree (Argania spinosa) is an endemic species from south-western Morocco. Argan-based preparations have been widely used in Moroccan traditional medicine for their biological properties, as well as for several cosmetic purposes. Whereas kernel, pulp of fruit and trunk have been extensively studied for their nutritional and pharmacological effects, relatively little is known about argan tree leaves.The main purpose of the present study is to investigate and characterise the bioactive phenolic fractions in both crude and aqueous extracts derived from argan tree leaves.A qualitative profile of the antioxidant phenolic compounds in argan leaves was obtained by means of structural hypothesis based on UV spectra and mass spectrometric fragmentation patterns. Moreover, selected phenolics were quantified in argan leaves by using a fully validated method based on liquid chromatography coupled to diode array detection and tandem mass spectrometry (LC-DAD-MS/MS). All the extracts were purified by a fast and reliable microextraction by packed sorbent (MEPS) procedure, before analysing them by LC-MS/MS.Based on retention times, mass spectrometric fragmentation and UV spectra, 13 phenolic compounds were identified or tentatively elucidated from crude and aqueous extracts derived from Argania spinosa leaves, while seven compounds were quantified in both extracts.The obtained results could represent a first step towards a complete characterisation of the argan plant, its bioactive profiling and the valorisation of its by-products as a source of potentially beneficial bioactive molecules.

MASSIVE OPEN ON-LINE COURSES IN OPEN EDUCATIONAL RESOURCES AND E-LEARNING FOR TOXICOLOGY COURSES Stefano GIROTTI1, Luca FERRARI2, Michele PROTTI1, Laura MERCOLINI1, Camelia DRAGHICI3 1Department of Pharmacy and Biotechnology FaBiT, Via San Donato 15, Alma Mater Studiorum - Università di Bologna, Bologna,40127, Italy 2Dipartimento di Scienze dell' Educazione 'G.M. Bertin', Alma Mater Studiorum - Università di Bologna, Via Filippo Re 6, 40126 Bologna, Italy 3Department of Product Design, Mechatronics and Environment, Transilvania University of Brasov, 29 Eroilor Blv, 500036 Brasov, Romania Remarkably, owing to the lack of European Massive Open On-line Courses (MOOCs) [1] in the field of the Toxicology and major differences in the teaching and learning of this important subject at various European biologically oriented faculties, we will present our TOX-OER European project which develops a scientific and pedagogical joint between research in the field of toxicology and MOOC pedagogical design [2]. This will consist in a guideline to support partners during: a) the creation of accessible Open Educational Resources (OER); b) course & module management; c) the implementation, monitoring and evaluation of individual and social learning activities. This procedure will contribute to the promotion of using the learning outcomes in the design and delivery of educational programs and activities in favor of pupils, students, young people, trainees, adult learners. Furthermore, the TOX-OER project could create the conditions for the RECOGNITION and CERTIFICATION (ECTS credits) of learning achievements, at least between partners. Finally, throughout the duration of the project, the partners involved in the educational tasks will manage a virtual space (server) within which the MOOC platform will be installed, and where all the Open Educational Resources will be available. TOX-OER project is coordinated by Universidad de Salamanca and partners are: Università di Bologna, Italy; Transilvania University of Brasov, Romania; Univerzita Karlova V Praze, Czech Republic; Universidade do Porto, Portugal; Space Research and Technology Institute, Bulgaria; Kymenlaakson Ammattikorkeakoululu Oy, Finland. [1] Chiappe, A., & Arias, V. (2015). Understanding reusability as a key factor for open education: A review. The International Review of Research in Open and Distributed Learning, 16(1). Retrieved from http://www.irrodl.org/index.php/irrodl/article/view/2042 ISSN: 1492-3831 [2] https://www.youtube.com/watch?v=U9lHtNLHP4Y

PB12. OVERCOMING BIOSAMPLING ISSUES IN SPORT DRUG TESTING: ANABOLIC STEROIDS IN DRIED URINES Michele PROTTI,1 Maria Carmen CATAPANO,1 Angelo E. SBERNA,2 Stefano GIROTTI,1 James RUDGE,3 and Laura MERCOLINI1 1Department of Pharmacy and Biotechnology (FaBiT), University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy 2 Sport Medicine, Enna Local Health Unit, Viale A. Diaz 7, 94100 Enna, Italy 3 Neoteryx LLC, 421 Amapola Avenue, 90501 Torrance, CA, USA Some crucial points are still under evaluation in the field of sampling and analysis for sport drug testing, including some limitations related to the biological fluid of choice: urine. Dried matrix (DM) collection is a very promising tool for improving anti-doping analysis, addressing those issues related to frequent and impromptu sampling, detection of substance concentrations, enhanced compound stability and need for rapid and high-throughput protocols. Ongoing innovations have demonstrated DM reliability, which is now directly applicable with the coupling to a wealth of microsampling techniques. This research involves in particular the use of dried urine spots (DUS) and volumetric absorptive microsampling (VAMS) on urines, for the collection of accurate volumes, followed by the LC-MS analysis of the main classes of the substances included in the World Anti-Doping Agency (WADA) prohibited list [1], with particular focus on compounds belonging to the class of anabolic steroids (testosterone, epitestosterone, dihydrotestosterone, androstenedione, methandrostenolone, mesterolone, nandrolone, norethandrolone and danazol. The goal of this project is to establish and validate simple but reliable protocols for the collection of urine microvolumes, unlikely to be tampered but transportable and, above all, storable with no precautions, to perform screening tests and targeted analysis according to the International Standard for Testing and Investigations (ISTI) and the International Standard for Laboratories (ISL), as part of the World Anti-Doping Code. These protocols would substantially reduce overall analysis costs, allowing their application not only to elite athletes, but also to amateurs in local laboratories. [1] “The World Anti-Doping Code: the 2016 Prohibited List”. World Anti-Doping Agency (WADA)

TOXICOLOGICAL OPEN EDUCATIONAL RESOURCE AND MOOCs (MASSIVE OPEN ON-LINE COURSES) IN E-LEARNING Stefano GIROTTI1, Luca FERRARI2, Michele PROTTI3, Laura MERCOLINI3 1Department of Pharmacy and Biotechnology, Via San Donato 15, University of Bologna, 40127 Bologna, Italy 2Department of Education Studies “G.M. Bertin”, University of Bologna, Via Filippo Re 6, 40126 Bologna, Italy 3Department of Pharmacy and Biotechnology, Via Belmeloro 6, University of Bologna, 40126 Bologna, Italy Introduction: Remarkably, owing to the lack of European MOOCs (massive open on-line courses) [1] in the field of the Toxicology and major differences in the teaching and learning of this important subject at various European faculties, we will present our Toxicological Open Educational Resource (TOX-OER) European project which develop a scientific and pedagogical joint between research in the field of toxicology and MOOC pedagogical design (i.e. a guideline to support partners during: a) the creation of accessible Open Educational Resources, b) course & module management, c) the implementation, monitoring and evaluation of individual and social learning activities) [2]. Materials and Methods: TOXOER project is coordinate by Universidad de Salamanca and partners are: Alma Mater Studiorum - Università di Bologna, Italy; Universitatea Transilvania din Brasov, Romania; Univerzita Karlova V Praze, Czech Republic; Universidade do Porto, Portugal; Space Research and Technology Institute, Bulgaria; Kymenlaakson Ammattikorkeakoululu Oy, Finland. Results and Discussion: Modules: 1-General Concepts; 2-Pharmaco-Toxicokinetics; 3-Principal Groups of Xenobiotics; 4-Environmental Pollutants; 5-Target Organ Toxicity and Biomarkers; 6-Environmental Toxicology; 7-Patents and Patent Application. Example of Script structure, 2 ECTS (56 hours total activity): - Textual introduction to the course -1x video introduction to the course [3 minutes] - 4x video lessons [15 minutes for each video] and/or commented slides [15 minutes for each video] - 4x intermediate test or active online learning activities (only when MOOC scenario releases a certificate of participation without legal value or ECTS credits) - 8x hours of further readings (text-based learning resources) - 1x final test (only when MOOC scenario releases a certificate of participation without legal value or ECTS credits) – Bibliography. Conclusion: This procedure will contribute to the promotion of using the learning outcomes in the design and delivery of educational programmes and activities in favour of students, trainees, pupils, adult learners and young people. Furthermore, the TOX-OER project could create the conditions for the RECOGNITION and CERTIFICATION (ECTS credits) of learning achievements, at least among partners. Finally, throughout the duration of the project, the partners involved in the educational tasks will manage a virtual space (server) within which the MOOC platform will be installed and where all the Open Educational Resources will be available. [1] Chiappe, A., & Arias, V. (2015). Understanding reusability as a key factor for open education: A review. The International Review of Research in Open and Distributed Learning, 16(1). Retrieved from http://www.irrodl.org/index.php/irrodl/article/view/2042 ISSN: 1492-3831 [2] https://www.youtube.com/watch?v=U9lHtNLHP4Y

OB5. NOVEL BLOOD AND URINE MICROSAMPLING STRATEGIES FOR THE MONITORING OF ALCOHOL CONSUMPTION MARKERS Laura MERCOLINI1, Michele PROTTI1, Maria C. CATAPANO1, Stefano GIROTTI1, Anna BARBIERI2, Laura SABATINI2, Vittorio BOIDO2, Vittorio LODI2, Francesco S. VIOLANTE2 1 Department of Pharmacy and Biotechnology (FaBiT), University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy 2 Department of Medical and Surgical Sciences (DIMEC), University of Bologna, Via P. Palagi 9, 40138 Bologna, Italy Ethyl glucuronide (EtG) and ethyl sulfate (EtS) are direct alcohol consumption markers, detectable in blood and urine even after consumption of trace amounts of ethanol. The detection window of these biomarkers is dose- and individual-dependent and ranges between 1 and 3 days after alcohol uptake, covering the time gap between short-term (e.g. ethanol) and long-term alcohol markers. Simultaneous determination of EtG and EtS could be, therefore, a suitable strategy for detection of recent alcohol consumption, with applicability in alcohol abstinence programs, workplace alcohol testing or as proof of alcohol uptake by court. However, the risk of bacteria presence in urine, that might hydrolyze or synthesize EtG and EtS and cause false identification of alcohol consumption, detracts from method reliability [1]. On the other hand, classic blood withdrawal via phlebotomy suffers from a number of inherent drawbacks: invasive sampling, conservation and transport requirements under controlled temperatures, the need to handle the samples immediately after collection (plasma separation) make this approach unappealing for application to large cohorts of subjects. The purpose of this study is the development of a new strategy for biosampling based on the use of dried matrix micro volumes: dried blood spots (DBS) and dried urine spots (DUS), followed by high-performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The application of these original methods for the determination of alcohol consumption markers will lead to standardized sampling, pretreatment and analysis protocols for a simple, reliable and high-throughput application for workplace alcohol testing. [1] A.H. Redondo, C. Körber, S. König, A. Längin, A. Al-Ahmad, W. Weinmann, Anal. Bioanal. Chem. 402, 2417-2424 (2012).

Anxiety is a complex psychiatric disorder with an unknown aetiology and involving several neurotransmitter systems. These constraints have meant that researchers have looked to develop drugs, which target a variety of molecular targets, with the aim of creating safer and more effective anxiolytic drugs. Apart from the 'traditional' GABAergic and serotonergic systems, the endocannabinoid, opioidergic, glutamatergic, neurokinin, and even cholinergic systems have been (and are being) considered as preferred targets for prospective new drugs.This review presents candidate drugs that were investigated for the treatment of anxiety-spectrum disorders and then discontinued between the 2009 and 2014 period.Despite the large variety of molecular targets, and the considerable financial and RD resources dedicated to finding treatment solutions for anxiety-spectrum disorders, a great number of candidates have failed to reach the market. Indeed, there is still an unmet need for more effective anxiolytics that give patients a better quality of life. Although there are inherent problems with psychiatric drug development, it is thought that repurposed drugs may provide some benefit in the future.

A new microextraction by packed sorbent (MEPS) procedure coupled to a high-performance liquid chromatographic method has been developed for quantitation of catecholamines (i.e. norepinephrine, epinephrine and dopamine) in innovative biological samples, namely dried plasma and urine spots. Analyses were carried out on a C18 reversed-phase column using a mobile phase composed of 2.5% methanol and 97.5% aqueous citrate buffer, containing octanesulfonic acid. Coulometric detection was used, setting the first analytical cell at −0.350 V and the second analytical cell at +0.400 V. Dried matrices were purified by means of a fast and feasible MEPS procedure, optimized on C18 sorbent and requiring only a small amount of biological sample. The availability of miniaturized procedures allowed satisfying specific requirements that ought to be considered during pre-treatment intended for catecholamine analysis. The extraction yield values were always higher than 85% and sensitivity was good, with a limit of quantitation of 100 pg mL −1 for all the analytes. Satisfactory results were also obtained in terms of linearity, precision and accuracy. The method was successfully applied to dried plasma and urine spots derived from two socially diversified groups, namely psychiatric patients and poly-drug abusers, in comparison to healthy volunteers.

An original HPLC-F method has been developed for the analysis of resveratrol and other antioxidants (cis-resveratrol, ferulic acid) as a first step in the study of the nutraceutical properties of grape-related products. The method takes advantages of the native fluorescence of the analytes (monitored at 386 nm, exciting at 298 nm) thus obtaining high sensitivity and selectivity. The pre-treatment consists of an innovative MEPS procedure, requiring very small amounts of sample and solvents. Under the chosen conditions, it was possible to quantify the analytes in several kinds of wine, as well as in must, in "grappa" (pomace brandy) and in the other matrices

Two original methods were developed and compared for TDM of topiramate: one based on HPLC with fluorescence detection and one based on CE with indirect UV detectio. Both analytical methods, fully validated, have shown to be suitable for the monitoring of patients undergoing therapy with topiramate.