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Since ancient times, Artemisia annua (A. annua) has been used as a medicinal plant in Traditional Chinese Medicine. In addition, recent studies have investigated the cytotoxic effects of A. annua extracts towards cancer cells. The leading aim of the present research is to evaluate the cytotoxic effects of an hydroalcoholic extract of A. annua on two canine osteosarcoma (OSA) cell lines, OSCA-8 and OSCA-40, focusing on the possible involvement of ferroptosis. The quantitative determination of artemisinin concentration in the extract, culture medium and OSA cells was carried out through the use of an instrumental analytical method based on liquid chromatography coupled with spectrophotometric detection and tandem mass spectrometry (LC-DAD-MS/MS). OSCA-8 and OSCA-40 were exposed to different dilutions of the extract for the EC

After 2 years of COVID-19 restrictions, the 8th Young Scientist Symposium was organized again as a face-to-face meeting covering a broad array of scientific presentations. As in the previous editions, the meeting was organized by young scientists for young scientists under the umbrella of the European Bioanalysis Forum and in collaboration with academia. The traditional Science Café was again included as an interactive round table session. This year, the main focus was on the challenges of communication. New for the 8th edition was a session connecting the young scientists with more seasoned experts in an effort to bridge talent and experience. In this article, we share the feedback of the scientific sessions and the Science Café held at the symposium.

The term seizures includes a wide array of different disorders with variable etiology, which currently represent one of the most important classes of neurological illnesses. As a consequence, many different antiepileptic drugs (AEDs) are currently available, exploiting different activity mechanisms and providing different levels of performance in terms of selectivity, safety, and efficacy. AEDs are currently among the psychoactive drugs most frequently involved in therapeutic drug monitoring (TDM) practices. Thus, the plasma levels of AEDs and their metabolites are monitored and correlated to administered doses, therapeutic efficacy, side effects, and toxic effects. As for any analytical endeavour, the quality of plasma concentration data is only as good as the analytical method allows. In this review, the main techniques and methods are described, suitable for the TDM of three AEDs belonging to the class of ion channel agents: ezogabine (or retigabine), lacosamide, and zonisamide. In addition to this analytical overview, data are provided, pertaining to two of the most important use cases for the TDM of antiepileptics: drug–drug interactions and neuroprotection activity studies. This review contains 146 references.

The 7th Young Scientist Symposium, a meeting again organized as a hybrid online event by young scientists for young scientists under the umbrella of the European Bioanalysis Forum and in collaboration with the Universities of Bologna and Ghent, included a variety of interesting presentations on cutting-edge bioanalytical science and processes. On the morning of day 2, the meeting hosted their traditional Science Café around the theme: ‘How has COVID-19 changed our future?’ in which the Young Scientist Symposium organizing committee engaged with the delegates on how the COVID-19 pandemic has impacted the careers of young scientists working in a bioanalytical (industry or academic) laboratory, that is, things they lost, for good or for bad – things they gained, wanted or unwanted, things they learned about themselves and their industry. This manuscript provides feedback from those discussions.

After the development of “classical” tricyclic antidepressants and monoamine oxidase inhibitors, numerous other classes of antidepressant drugs have been introduced onto the market. The selective serotonin reuptake inhibitor class is the best-known one, but many others exist, usually identified by their mechanism of activity. In this second part of the review, focused on new-generation antidepressants not included among selective serotonin reuptake inhibitors, the following classes are considered: noradrenergic and selective serotonergic antidepressants; norepinephrine reuptake inhibitors; serotonin, norepinephrine and dopamine reuptake inhibitors; melatonergic agonists and selective serotonergic antagonists; norepinephrine and dopamine reuptake inhibitors; and so forth. These different mechanisms underlie tolerability and safety profiles that can be very different among the classes, with each one providing significant advantages and disadvantages in comparison with others. The main characteristics of the following antidepressants are described: mianserin, mirtazapine, setiptiline, reboxetine, viloxazine, teniloxazine, atomoxetine, nefazodone, agomelatine, bupropion, esketamine, and tianeptine. The paper is focused on their metabolism and interactions, but also includes brief notes on analytical methods useful for their therapeutic drug monitoring.

Vitamin K is traditionally connected with blood coagulation, since it is needed for the posttranslational modification of 7 proteins involved in this cascade. However, it is also involved in the maturation of another 11 or 12 proteins that play different roles, encompassing in particular the modulation of the calcification of connective tissues. Since this process is physiologically needed in bones, but is pathological in arteries, a great deal of research has been devoted to finding a possible link between vitamin K and the prevention of osteoporosis and cardiovascular diseases. Unfortunately, the current knowledge does not allow us to make a decisive conclusion about such a link. One possible explanation for this is the diversity of the biological activity of vitamin K, which is not a single compound but a general term covering natural plant and animal forms of vitamin K (K1 and K2) as well as their synthetic congeners (K3 and K4). Vitamin K1 (phylloquinone) is found in several vegetables. Menaquinones (MK4-MK13, a series of compounds known as vitamin K2) are mostly of a bacterial origin and are introduced into the human diet mainly through fermented cheeses. Current knowledge about the kinetics of different forms of vitamin K, their detection, and their toxicity are discussed in this review.

Herein it is reported the development and application of two chromatographic assays for the measurement of the activity of 3-Hydroxyanthranilate-3,4-dioxygenase (3HAO). Such an enzyme converts 3-Hydroxyanthranilic acid (3HAA) to 2-amino-3-carboxymuconic semialdehyde (ACMS), which undergo a spontaneous, non-enzymatic cyclization to produce quinolinic acid (QUIN). The enzyme activity was measured by quantitation of the substrate consumption over time either with spectrophotometric (UV) or mass spectrometric (MS) detection upon reversed-phase chromatographic separation. MS detection resulted more selective and sensitive, but less accurate and precise. However, both methods have sufficient sensitivity to allow the measurement of enzyme activity with consistent results compared to literature data. Since MS detection allowed less sample consumption it was used to calculate the kinetics parameters (i.e., Vmax and Kd) of recombinant 3HAO. Another MS-based method was then developed to measure the amount of QUIN produced, revealing an incomplete conversion of 3HAA to QUIN. As suggested by previous studies, the enzyme activity was apparently sensitive to the redox state of the enzyme thiols. In fact, thiol reducing agents such as dithiothreitol (DTT) and glutathione (GSH), can alter the enzyme activity although the investigation on the exact mechanism involved in such effect was beyond the scope of the research. Interestingly, edaravone (EDA) induced an in vitro suppression of QUIN production through direct, competitive 3HAO inhibition. EDA is a molecule approved for the treatment of amyotrophic lateral sclerosis (ALS), a neurodegenerative disease associated with an increase of QUIN concentrations in both serum and cerebrospinal fluid. Although EDA was reported to mitigate ALS progression its mode of action is still largely unknown. Some studies reported antioxidant and radical scavenger properties of EDA, but none confirm a direct activity as 3HAO enzyme inhibitor. Since QUIN is reported to be a neurotoxic metabolite, 3HAO inhibition can contribute to the beneficial effect of EDA in ALS, although such a mechanism must be then confirmed in vivo. However, EDA might be a convenient scaffold for the design of selective 3HAO inhibitors with potential applications in ALS treatment.

Metabolic syndrome is a complex pathology characterized by imbalances in lipid and glucose metabolism and weight gain, and consequently by an increase in the incidence of type II diabetes and cardiovascular disease. Metabolic syndrome is rapidly becoming one of the most important side effects of treatment with modern “atypical” antipsychotic agents, probably due to their specific mechanisms of action. Although the most recent members of this class (aripiprazole, asenapine, ziprasidone) seem to produce a reduce incidence of metabolic syndrome, the problem is far from being resolved. In this chapter, the three most important classical neuroleptics (chlorpromazine, haloperidol and loxapine) and the most important atypical antipsychotics will be individually analyzed in relation to their propensity to cause metabolic syndrome. The most reliable, current data will be presented, also in the perspective of possible interventions to mitigate metabolic imbalances, comparative studies, switching studies and augmentation strategies. An important strategy for metabolic syndrome prevention could also be the performing of an accurate therapeutic drug monitoring (TDM). Thus, an up-to-date overview will also be presented of recent and significant analytical methods for the determination of the drugs of interest and their main metabolites in human biological fluids.

Since 2014, the European Bioanalysis Forum (EBF) organizes a Young Scientist Symposium. The meeting format was created to provide development opportunities for young scientists to engage in international discussions. Creating a peer community of young scientists has been a proven recipe to lower the threshold and promote engagement in this community of young talents. At the same time, the meetings are aimed at stimulating collaboration between the EBF and academia. This manuscript summarizes the discussions at the Fifth EBF Young Scientist Symposium, held in Bologna (Italy) between 20 and 21 March 2019.

The objective of this study was to incorporate wild garlic (A. ursinum) extract into microparticles (MPs) in order to protect its valuable active compounds and improve its oral bioavailability. For this purpose, spray congealing technology was applied and Gelucire 50/13 (Stearoyl polyoxyl-32 glycerides) was selected as MPs carrier. MPs were characterized in terms of yield, encapsulation efficiency and particle size. Differential scanning calorimetry (DSC) and Fourier-transform infrared spectroscopy (FT-IR) analysis of MPs showed the absence of chemical interactions between carrier and extract and suggested that spray congealing process did not modify nor degrade the encapsulated extract. The encapsulation into MPs led to an improvement of the extract dissolution performance as well as an enhancement in solubility of > 18 fold compared to the pure extract. Additionally, MPs were stable over three months showing only a minor decrease in the content of active compounds (allicin and Smethyl methanethiosulfonate) and maintaining a good antimicrobial activity. Therefore, obtained results suggested that the encapsulation of A. ursinum extract in MPs by spray congealing is a promising approach to improve the biopharmaceutical properties of the extract, without affecting its antibacterial activity.

Volumetric absorptive microsampling (VAMS) is a recent microsampling technique used to obtain dried specimens of blood and other biological matrices for application to a plethora of bioanalytical purposes. As such, it can be likened to dried blood spot (DBS) technique that has been in wide use for the last 40 years. However, VAMS promises to bring some significant advantages over DBS, related to sampling volume accuracy, haematocrit (HCT) dependence, pre-treatment and automation. Although some aspects still need to be investigated in depth, VAMS is increasingly recognised as a viable alternative to DBS and other dried microsampling techniques. In this tutorial, different aspects of VAMS approach are described and discussed, presenting the procedures adopted and the results obtained by those authors who have developed this kind of analytical workflow in the last few years. Hopefully, this will help other scientists to find new solutions to old and recent problems related to microsampling and to produce new, sound and interesting science in this field.

Due to the lack of European Massive Open Online Courses (MOOCs) in the field of the Toxicology and the major differences in the styles of teaching and learning of this important subject in amongst various European life science-oriented institutions, the European Erasmus+ project “Learning Toxicology through Open Educational Resources (TOX-OER)” was developed and implemented. Considering the complexity and heterogeneity of the toxicology field, the TOX-OER project main objective was to develop and share toxicology-related knowledge and skills among students/ earners from seven countries, which promote the internationalization of Higher Education Institutions in Europe but also in countries from other continents. The project was coordinated by Universidad de Salamanca (Spain), and the partners were: Space Research and Technology Institute (Bulgaria), Univerzita Karlova V Praze (Czech Republic), South-Eastern Finland University of Applied Sciences (Finland), Università di Bologna (Italy), Universidade do Porto (Portugal) and Universitatea Transilvania din Brasov (Romania). One of the goals/ objectives of the project is the dissemination, popularizing the information and reaching potentially interested people who can benefit from the offered courses. The aim of this paper is to present and analyze part of the TOX-OER’s outcomes developed by the project’s partnership, especially the modules/ topics related to the drugs, gaseous and persistent organic pollutants, as the principal groups of xenobiotics.

Paper-based sorptive phases (PSPs) are functional planar materials with a demonstrated potential in analytical sample preparation. This article describes the synthesis of a polyimide coated paper by an in-situ imidization at a high temperature. Polyimides (PI) are synthesized in two subsequent steps where a hydrophilic polymer, in this case, poly(amic acid) (PAA), is formed as an intermediate product. PAA is finally transformed into hydrophobic PI by thermal curing at 180 °C. The synthesis of PI-paper takes advantage of this two-step procedure. In the first stage, a segment of filter paper is immersed into an aqueous PAA solution. After the solvent evaporation, the paper is heated at 180 °C for 1 h inducing the formation of the hydrophobic PI over the cellulose fibers. Infrared spectroscopy has been used to characterize the synthesized materials by defining a coverage factor F. The hydrophobicity of the materials has been studied using an aqueous methylene blue solution as a marker. To fully demonstrate the usefulness of the material in the sample preparation field, the extraction of methadone from oral fluid (OF) samples has been considered as a model analytical problem. The main variables affecting the synthesis (PAA concentration on the precursor solution and number of dips) and the extraction (elution and extraction times) have been fully evaluated. Working under the optimum conditions, a limit of quantification of 9 µg/L, intraday and interday precision better than 14.6%, and accuracy in the range of 87-108% were obtained.

An original, innovative, high-throughput method based on attenuated total reflectance - Fourier’s transform infrared (ATR-FTIR) spectroscopy has been developed for the proof-of-concept discrimination of fibre-type from drug-type Cannabis sativa L. inflorescences. The cannabis sample is placed on the instrument plate and analysed without any previous sample pretreatment step. In this way, a complete analysis lasts just a few seconds, the time needed to record an ATR-FTIR spectrum. The method was calibrated and cross-validated using data provided by liquid chromatography - tandem mass spectrometry (LC–MS/MS) analysis of the different cannabis samples and carried out the statistical assays for quantitation. During cross-validation, complete agreement was obtained between ATR-FTIR and LC–MS/MS identification of the cannabis chemotype. Moreover, the method has proved to be capable of quantifying with excellent accuracy (75–103 % vs. LC–MS/MS) seven neutral and acidic cannabinoids (THC, THCA, CBD, CBDA, CBG, CBGA, CBN) in inflorescences from different sources. The extreme feasibility and speed of execution make this ATR-FTIR method highly attractive as a proof-of-concept for a possible application to quality controls during pharmaceutical product manufacturing, as well as on-the-street cannabis controls and user counselling.

Vitamin A is a group of vital micronutrients widely present in the human diet. Animal-based products are a rich source of the retinyl ester form of the vitamin, while vegetables and fruits contain carotenoids, most of which are provitamin A. Vitamin A plays a key role in the correct functioning of multiple physiological functions. The human organism can metabolize natural forms of vitamin A and provitamin A into biologically active forms (retinol, retinal, retinoic acid), which interact with multiple molecular targets, including nuclear receptors, opsin in the retina and, according to the latest research, also some enzymes. In this review, we aim to provide a complex view on the present knowledge about vitamin A ranging from its sources through its physiological functions to consequences of its deficiency and metabolic fate up to possible pharmacological administration and potential toxicity. Current analytical methods used for its detection in real samples are included as well.

The 6th Young Scientist Symposium, a meeting organized by young scientists for young scientists under the umbrella of the European Bioanalysis Forum vzw and in collaboration with the Universities of Bologna and Ghent, included a variety of interesting presentations on cutting edge bioanalytical science and processes. Integrated in the meeting, an interactive round table session, the Science Café, discussed the challenges related to sustainability for bioanalytical lab activities. This manuscript reflects conclusions from these discussions. They can provide our community a compass for future business practices to embrace more sustainable laboratory activities considerate of smarter use of a wide array of resources and laboratory tools, resulting in increased wellbeing for our next generations and our planet.

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.

As part of the European Bioanalysis Forum mission to provide development opportunities for scientists, a Young Scientist Symposium has been organized every year since 2014. The meetings, organized by and for young scientists, aim at immersing talent from industry and academia in the scientific and process challenges important for their (future) professional environment. In an ideal world, the setting of an interactive symposium in stimulating auditorium sets the foundation for long lasting peer scientific relationship. This year, a pandemic has descended across all continents, changing the dynamics of the meeting. This manuscript summarizes the discussions at the Sixth EBF Young Scientist Symposium, originally planned as a face-to face event in March 2020 in Bologna, Italy but finally executed as a hybrid meeting in Cyberspace and on location in a few regions across Europe between 24–25 September 2020.

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.

Objective: Therapeutic Drug Monitoring (TDM) is an evidence-based practice consistent with the assumption that pharmacological plasmatic concentrations correlate better with clinical effects than prescribed doses of the used drugs. Considering the limited efficacy of Eating Disorders (ED)’s pharmacological treatment and the high rate of adverse effects, TDM may represent a valid tool to check antidepressant drugs (AD) that are frequently prescribed in patients with ED. We aim to test the feasibility of TDM in a real-life setting and to understand the reliability of oral fluid measurements related to whole blood ones. Methods: We enrolled patients attending the ED outpatient clinic in Bologna with a Body Mass Index (BMI) 30 kg/m2 treated with antidepressants. We collected oral fluid samples and whole blood dried microsamples by finger puncture using VAMS (Volumetric Absorptive Microsampling) technique from patients to determineTDM. Results: Nineteen patients participated in our study. Participants were treated with Sertraline (N=5), Fluoxetine (N=6), Vortioxetine (N=4), Citalopram (N=2), Escitalopram (N=1), Fluvoxamine (N=1). Preliminary results by our pilot research report show a positive correlation between plasmatic and salivary concentrations only for Vortioxetine. Additionally, only the Vortioxetine shows a pattern of plasmatic concentrations independent from the volume of distribution at the steady-state, even though the wide range of BMI. Conclusions: Despite the small size of the sample, by considering these preliminary data, we are confident that further studies will allow us to outline that TDM may represent a valid tool to achieve a consistent clinical efficacy of AD in patients diagnosed with ED.

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.