Bioanalysis is a subdiscipline of analytical chemistry for the identification and quantitative measurement of xenobiotics and biotics in biological matrices. Many scientific decisions regarding drug development, drug dosing and patient safety are dependent upon the accurate quantification of drugs and endogenous components in biological samples.

Yu and Bashaw, Bioanalysis (2014) 6(4), 505-510 DOI: 10.4155/bio.13.334

One very unique feature of contemporary bioanalysis is that its measurement target is at very low concentration levels (typically at low ng/mL and even at pg/mL). It is this very low concentration that challenges bioanalytical scientists to accurately measure the analytes of interest. This is compounded by coexisting endogenous or exogenous compounds with similar chemical structures to the target analytes at a much higher concentration.

Over the past 30 years, drugs have become more and more powerful and only advances in technology have allowed the measurements of drugs and their metabolites at the lower circulating concentrations. At the same time, the users of the drug concentration data have asked for data with improved accuracy and precision to enable the development of better mathematical models that can support the approval of drugs. To ensure patient health and safety, the response of health authorities has been to increase their requirements regarding the quality and the integrity of bioanalytical results in bioanalytical validations and in the analysis of both non-clinical and clinical study samples for drugs and metabolites.

A lot has happened in recent years to foster faster, cheaper, and better ways of providing quality bioanalytical results. Various agencies from a number of countries and regions including Brazil, Canada, China, European Union, India, Japan and the United States have made collective efforts in different periods to regulate and harmonize their guidance on bioanalytical method development and validation, with the ultimate goal of improving the quality of bioanalytical results also considering the characteristics of the analytical methods used in bioanalysis, e.g., chromatographic assay and ligand binding assay.

The International Council for Harmonization, in the ICH M10 guideline, has combined the current regional guidelines/guidance and related documents, in order to avoid differences in terminology, reduce the need for additional validation experiments of being compliant with multiple guidelines and support streamlined global drug development.

In my presentation, the attention is focused on regulatory and practical perspectives with particular regard to the terminology and the main parameters to be evaluated during a validation process of a bioanalytical method.

Capillary electrophoresis in its different versions represents a powerful separation technique which proved sufficiently competitive/complementary to high performance liquid chromatography. Based on the simultaneous action of electromigration and electroosmotic flow, this approach offers a wide range of conditions under which successful separations and quantitative determinations can be obtained. In addition to the classical separation mode based on typical electrophoretic conditions, other separation modes have been successfully applied in capillary format, including micellar electrokinetic chromatography, electrochromatography, isoelectrophocusing, gel electrophoresis, isotacophoresis etc. On the detection side, the miniaturized flow of fluid generated allows the adoption of different detection modes, including UV absorption, fluorescence, and mass spectrometry. Since in forensic analysis we are constantly faced with complex biological matrices in which a wide range of analytes (spanning from inorganic ions to small drugs, peptides, proteins and DNA fragments), the versatility of capillary electrophoresis makes it extremely attractive, at least as a confirmatory technique.

In the present lecture, selected examples of applications will be discussed with particular attention paid to ion analysis, chiral separations and determination of proteins of forensic interest.

Schematic drawing of capillary electrophoresis instrumentation. Upon applying voltage on the capillary, separation occurs according to the electric charge/molecular mass of the ions: anions migrate to anode, cations to cathode. The electroosmotic flow moves the whole content of the capillary from anode to cathode (in most cases).

Bioanalytical method validation is a mandatory step to evaluate the ability of developed methods to provide accurate results for their routine application in sample analysis. Method validation performed before sample analysis relies on the use of spiked biological matrix samples (QCs) to mimic the real samples in the assessment of precision, accuracy and stability. The assumption is that if the method meets acceptance specifications as measured by the QC samples then the results obtained for the study samples are valid and can be reported with confidence for decision making.

Nevertheless, even fully validated and well characterized methods have a degree of uncertainty since not all the issues faced in study samples analysis can be anticipated. There are two major types of issues that could arise during sample analysis: previously validated parameters that need further refinement/adjustment and unexpected events that did not occur during method validation. The first type of issues includes for instance the validation of a higher dilution factor to bring the concentration of a set of samples within the calibration range or the evaluation of analyte stability to address a sample storage/handling problem. The latter type includes selectivity or variation of response (matrix effect) due to unintended analytes, unknown metabolites and interfering compounds. The reason why these phenomena are not observed in pre-study method validation is that spiked QC samples can ’t account for metabolic processes and all chemical transformations that take place in living organisms.

This presentation is aimed to give an overview of in-study validation through the discussion of select cases and to provide some tips for managing unforeseen issues in sample analysis.

The MIST strategy is a hot topic of drug development process and nowadays is strongly recommended by the regulatory agency to ensure that the safety of metabolites found in humans is evaluated adequately in toxicological studies. The regulatory agencies encourage an early understanding of the human metabolism to ensure that human circulating metabolites above a defined threshold are also observed in preclinical species at equal or greater levels than in human. Moreover, it is also suggested to evaluate the potential contribution of relevant metabolites when conducting DDI risk assessment. The main challenge is how to balance the front-loading of metabolism studies with the need to appropriately invest resources according to the stage of drug development. A suggested option is to adopt ”alternative approaches”, early in the drug development process, able to generate reliable data for enabling prompt and informed decisions.

This presentation will cover various ”alternative approaches” for an early evaluation of the major human circulating metabolites and for the assessment of their toxicological cover in preclinical species, in the absence of synthetic metabolite reference standards before performing a Human Radiolabelled Study.

The term ”bioanalysis” is generally used to describe the quantitative determination of drug(s) and its metabolites in biological matrices. The bioanalytical goal is to develop a reliable, rapid and effective method for the determination of pharmacokinetic, pharmacodynamic and toxicokinetic parameters. Liquid chromatography/Mass Spectrometry is the preferred and commonly used tool in bioanalytical laboratories for quantitative biological sample analyses. Of course, clinical sample analysis support requires high level of regulatory and ethical principles with the ultimate goal of improving the health and welfare of patients and advancing biomedical science. For these reasons, a GCP standard approach has to be applied during all critical sample analysis steps in a clinical trial. In particular, plasma sample analysis activity has to be performed following the regulatory agency guidelines in order to keep under control both scientific and safety aspects. Key topic to check during the clinical sample analysis are, for example, the instruction for the collection, temporary storage and shipment of the samples, the conditions of receipt, the number of the samples that can be analysed within one batch of analysis, the rapid turnaround of the data release (crucial aspect to support dose escalation studies and if an adverse event occurs during clinical treatment), the primary and secondary endpoints of the clinical protocol, samples analysis authorization and confidentiality (consequently what is reported in Informed Consent document and the compliance with GDPR are fundamental). Moreover, as per regulatory point of view, the incurred samples reanalysis (ISR) has to be applied at least to all pivotal comparative BA/BE studies, first clinical trial in subjects, first patient trial, first trial in patients with impaired hepatic and/or renal function. The ISR assessment evaluates the robustness of the bioanalytical method during the real samples analyses, since the calibration standard and quality control samples used to perform the method validation may not mimic the real samples behaviour. Samples could differ by different protein binding, back-conversion of known and/or unknown metabolites and other several factors. If this assessment fails, an investigation including more corrective actions has to be planned and performed, in order to be sure that the concentration levels obtained from the real samples are accurate and precise.

References
1. EMEA/CHMP/EWP/192217/2009 Rev.1 Corr.2**, Last updated 03 June 2015;
2. ICH: Bioanalytical Method Validation M10 Draft version - Step 2b EMEA/CHMP/ICH/172948/2019;
3. Food and Drug Administration, Center for Drug Evaluation and Research (CDER), Center for Veterinary Medicine (CVM), May 2018;
4. Reflection paper for laboratories that perform the analysis or evaluation of clinical trial samples. EMA/INS/GCP|532137|20I0: Feb 2012;
5. ICH Topic E6 (R1) guidelines for Good Clinical Practice July 2002 CPMP/ICH/135/95;
6. EMA/CVMP/EWP/16/2000: Guideline on the investigation of bioequivalence.

Metabolomics, the most analytical of ”omics” technologies, is leading to a growing number of mainstream biomedical applications. In particular, metabolomics is increasingly being used to diagnose disease, understand disease mechanisms, identify novel drug targets, customize drug treatments and monitor therapeutic outcomes.¹

We will describe the bases of metabolomics, focusing on the application of metabolomics towards uncovering the underlying causes of complex diseases. A particular interest has been reserved to evaluate the metabolic changes in neoplastic cells to identify new diagnostic strategies and therapeutic targets.

As an example, the metabolic changes present in neuroendocrine tumors (NETs) have not been well characterized. Our aim was to perform a metabolic characterization of gastrointestinal (GI) NETs patients included in the phase II/III randomized placebo controlled multicentric and doble-blind AXI-IIG-02 trial, in order to better understand the tumoral metabolic alterations and trying to identify new therapeutic approaches to personalize the treatment of metastatic NETs patients. Plasma samples were profiled by GC-MS, CE-MS and LC-MS untargeted metabolomics. OPLS-DA was performed to evaluate metabolomic differences. Related pathways were explored using Metaboanalyst 4.0. Finally, ROC and OPLS-DA analyses were performed to select metabolites with biomarker potential. We identified 155 differential compounds between NETs and controls. We have detected an increase of bile acids, sugars, oxidized lipids and oxidized products from arachidonic acid and a decrease of carnitine levels in NETs. MPA/MSEA identified 32 enriched metabolic pathways in NETs related with the TCA cycle and amino acid metabolism.

This study provides a comprehensive metabolic profile of NET patients and identifies a distinctive metabolic signature in plasma of potential clinical use.

References
1. Wishart, D. Emerging applications of metabolomics in drug discovery and precision medicine. Nat Rev Drug Discov 15, 473-484 (2016). DOI: 10.1038/nrd.2016.32
2. Soldevilla B, López-López A, ... Barbas C, Garcia-Carbonero R. Comprehensive Plasma Metabolomic Profile of Patients with Advanced Neuroendocrine Tumors (NETs). Diagnostic and Biological Relevance. Cancers (Basel). 2021 May 27;13(11):2634. DOI: 10.3390/cancers13112634

During drug development processes, gaining insights into the lead compound's pharmacokinetic and pharmacodynamic properties is crucial. When it comes to the treatment of brain diseases, collecting information at the site of action is challenging. Cerebrospinal fluid (CSF) concentrations do not necessarily reflect the real concentration in the brain parenchyma. Drug concentrations in the CSF give information regarding drug transport across the choroid plexus, which is the main component of the blood-CSF barrier, but such concentrations do not provide information concerning blood-brain barrier (BBB) transport. This misconception hinders progress in the development of drugs targeting the central nervous system, as explained by Pardridge. Apart from determining exogenous drug concentrations for pharmacokinetic and pharmacodynamic studies, a fundamental part of central nervous system drug development is the monitoring of endogenous molecules at the site of action to gain insights into the physiology of the brain and its disease processes.^1-2 In fact, there are only a few techniques available that allow for the direct sampling from the cerebral interstitial space and thus provide insight into real concentrations in the brain parenchyma. Such techniques are microdialysis and cerebral open flow microperfusion (cOFM). Initially, these techniques were developed to monitor neurotransmitters and other small molecules, but over the years advances in the field have made it possible to monitor macromolecules (neuropeptides, proteins, ...) as well.^1,3-4

The first part of my PhD research was connected to a project wherein brain-penetrating nanobodies were generated with the final aim to circumvent the BBB for delivery of therapeutic biologics to the central nervous system.⁵ The overall aim of my work package in this project was to optimize a sampling technique in order to gain insight into the brain-penetrating properties and the pharmacokinetic profile of these nanobodies at their site of action. Therefore, we assessed the applicability of cOFM and microdialysis (AtmosLM^TM, 1 MDa cut-off) as techniques to sample interstitial fluid macromolecules (±31 kDa) in an acute setting. Performance of both methods was compared by evaluating the neuroinflammatory response, the BBB integrity, the in vitro recoveries and in vivo AUCs.⁶

The presentation will focus on the second part of my PhD. Here, we apply cOFM to sample interstitial fluid macromolecules in a chronic setting. More specifically, we want to gain insight into neurofilament light chain (NfL; 68 kDa) levels in the kainic acid mouse model for temporal lobe epilepsy (TLE) during the various stages of the disease model. NfL is a protein, solely located in the neuronal cytoskeleton and released into the interstitial fluid of the brain upon axonal injury and neurodegeneration.⁷ In this part of the project, cOFM is especially an interesting technique. Apart from the open exchange area with macroscopic openings, the limited neuroinflammatory response allows for repeated sampling and therefore long-term monitoring of NfL levels is possible within the same mouse in the chronic TLE model.

Research in the field of epilepsy remains of utmost importance. Every year, approximately 2.4 million people are diagnosed with epilepsy. The most common form, developed by structural causes such as traumatic brain injury, is TLE, which is difficult to control by antiseizure drugs and is often associated with severe comorbidities. The kainic acid mouse model for TLE is particularly clinically relevant because, similar to TLE patients, it induces typical cellular changes in the hippocampus, such as loss of inhibitory and excitatory neurons, dispersion of the dentate gyrus and extensive astrogliosis. Likewise, this chronic model is achieved by an initial event, unilateral kainic acid injection inducing a status epilepticus, and mice start developing spontaneous limbic seizures after a latent period of ±1 week. Thereafter, mice display a stable amount of spontaneous hippocampal seizures, which electrographically resemble the hippocampal seizures seen in TLE patients.⁸ The aim of this experiment is to investigate the effect of status epilepticus and of chronic recurrent spontaneous seizures (up till 5 weeks) on hippocampal interstitial fluid NfL levels using cOFM. cOFM probes are implanted in the ventral hippocampus of C57BL/6J mice. Status epilepticus is induced by unilateral dorsal intrahippocampal kainic acid injection. The use of both a single molecule array and enzyme-linked immunosorbent assay was explored for sample analysis. To obtain proper translational value for disease progression as well as treatment response, we will also assess the correlation with NfL CSF and plasma levels, and the effects of adequate status epilepticus suppression on the NfL levels using diazepam. This is especially interesting, since in a clinical set-up this would be common practice. Feasibility to sample interstitial fluid NfL using cOFM was first verified. Basal interstitial fluid NfL levels of 10-12-week C57Bl/6J mice are 3.02 ± 0.32 ng/mL (n = 5). The experiments in the model for TLE are currently ongoing. Preliminary results suggest that ISF NfL levels tend to increase during status epilepticus but not during the chronic phase with spontaneous recurrent seizures.

Overall, the reliable quantification of interstitial NfL using cOFM could be of interest for evaluating the disease activity, facilitating treatment development or monitoring of treatment responses in future experiments.

References
1. Custers M.-L., Nestor L., De Bundel D., Van Eeckhaut A., Smolders I. Pharmaceutics 2022, 14. 2. Pardridge W.M. Nat. Rev. Drug Discov. 2002, 1: 131-139.
3. Birngruber T., Sinner F. Drug Discov. Today Technol. 2016, 20: 19-25.
4. Hammarlund-udenaes M. AAPS J. 2017, 19: 1294-1303.
5. Wouters Y., Jaspers T., De Strooper B., Dewilde M. Fluids Barriers CNS 2020, 17: 4-13.
6. Custers M.-L., Wouters Y., Jaspers T., De Bundel D., Dewilde M., Van Eeckhaut A., Smolders I. Anal. Chim. Acta 2021, 1178: 338803.
7. Lambertsen K.L., Soares C.B., Gaist D., Nielsen H.H. Brain Sci. 2020, 10: 1-29.
8. Pitkanen A., Ekolle Ndode-Ekane X., Lapinlampi N., Puhakka N. Neurobiol. Dis. 2019, 123: 42-58.

Abruzzo region is characterized by a high degree of biodiversity, reflected also by a great number of endemic plant species (more than 3200).¹ In particular, many of these latter are used in the liquor industry which in Abruzzo boasts a centuries-old tradition. Final products obtained from the extraction of wild and / or cultivated herbs are in most cases deprived of a trademark of typicality, like DOP, DOC, or IGP. Thus, liquor production is often entrusted to handcrafted recipes handed down by generation after generation without an agronomic, a phytochemical, and a processing peculiar characterization in general. Hence the need to undertake specific studies in order to standardize the whole production process of liquors typical of Abruzzo region is still desirable. In particular, emphasis on the maximization of extractive yields concerning phytochemicals responsible for the organoleptic properties of alcoholic beverages and assessment of their chemical fingerprint for quality control purposes is totally missing.

The project of the Doctorate Course has been first focused on the development of optimized extraction and analytical sample preparation processes of the natural compounds responsible for the organoleptic properties of the final product. The quality of both processes has been examined by chromatographic (HPLC, UPHLC, GC-MS) and spectroscopic (UV, FLD, FT-IR, NMR) techniques. In recent years the trend in the development of new chemical processes in general is to frame the various methodologies in an environmentally friendly context, in order to minimize the impact of the extraction procedure on the environment, recycling and reusing solvents and materials selected for the purpose, as well as to gain an easier reproducibility of the listed methods for pilot plant and industrial scale usage.

With reference to the extraction processes applied in the herbal sector, a first element of innovation is represented by the use of extraction methods alternative to the well-known maceration, decoction, Soxhlet and similar conventional techniques. As widely reported in the recent literature, processes promoted by ultrasound and microwaves or by the addition of auxiliary extraction agents as ionic liquids, cyclodextrins, or inorganic and organic solids supports, for a selective adsorption, allow to significantly increase the extraction yields (on average of one factor between 3 and 10), improving the characteristics of the chemical fingerprint of the extracts.²

Currently, the project has reached the step of experiments performed and optimized using the following plant matrices of great economic value: Gentiana lutea (yellow gentian), Artemisia spp., Punica granatum (pomegranate), Rheum palmatum (rhubarb), Cassia angustifolia (senna) for the liquor productions, and Crocus sativus (saffron), Curcuma longa (turmeric) and Rhamnus frangula (buckthorn) for pharmaceutical and nutraceutical purposes.

H₂O, EtOH, and hydroalcoholic extracts were investigated in deep details with the aim to selectively isolate naturally occurring biologically active secondary metabolites belonging to different classes like anthraquinones, coumarins, flavonoids, phenolic acids, and secoiridoids, which are all featured by notable therapeutic and healthy properties, as well as valuable sensorial and organoleptic ones. Therefore, the main purposes of the studies accomplished during the PhD course were to investigate the possibility of coupling innovative and chemically green extraction techniques like solid phase extraction (SPE) with the most common analytical hyphenated techniques to reveal the effective and selective extraction of the above-mentioned phytochemicals.

For the SPE key step, we evaluated a panel of 21 biocompatible differently structured solid sorbents. Typically, the mostly used solid supports to accomplish SPE are represented by activated charcoal, silica gel, alumina, polymers, resins,³ and nanosized materials. In particular these latter, thanks to their absorption kinetics and their wide surface area, are currently widely used for the purpose as well.⁴ Nevertheless, all the listed sorbents have some limitations (costs, tedious and hard to handle synthesis, no possibilities of an effective recycle, etc.). Hence the need for new, more versatile, alternative, and efficient materials for SPE purposes is still alive and has a great and more and more growing interest.

In this context, layered double hydroxides (LDH), also known as "hydrotalcites" or "synthetic clays" having a general formula Mg₆Al₂(OH) ₁₆CO₃ 4H₂O and a typical lamellar structure, are nowadays considered as an attractive class of inorganic or mixed inorganic / organic solids possessing positively charged layers of transition and/or alkaline earth metal hydroxides counterbalanced by numerous anions, and may host H₂O molecules in the interlayer spaces. LDHs have several industrial applications to produce a plethora of cosmetic, nutraceutical, and pharmaceutical preparations.⁵ In fact, numerous drugs can nowadays be delivered by hydrotalcites, and several of these preparations are currently used in therapy.

Along with LDHs, magnesium oxide and hydroxide, and the phyllosilicates talc, different types of mica and bentonite were also selected for our studies. Advantageous characteristics to hypothesize a selective extraction of secondary metabolites from the above-mentioned plant matrices, are represented by the intimate 3D structure of such materials. Indeed, the phyllosilicates' chemical structure is based on six member rings of SiO₄4- tetrahedra interconnected in sheets. Three out of the 4 oxygens from each tetrahedra are shared with another one. This leads to a basic structural unit of Si₂O₅2-. Since most of them contain a hydroxyl ion, located at the center of the six membered rings, the structure becomes Si₂O₅(OH)3-. The -OH ions can create interactions with other cations (majorly Fe2+, Mg2+ and Al3+) that might be bonded to the SiO₄4- sheets, creating an octahedral coordination. Finally, the brucite-like structure of MgO and Mg(OH) ₂ composed by octahedral layers render such solids very similar to the LDHs.

The general experimental route set up and optimized in our laboratories implied that each sample extract is dissolved and / or suspended in double-distilled H2O and divided into different aliquots, each of them then treated with the corresponding solid support. All the mixtures were kept under magnetic stirring at room temperature for 24h and finally filtrated under vacuum. The filtrates were then analyzed, and the phytochemicals of interest quantified by HPLC-DAD. The quantification was evaluated first by calculating the differences in concentrations of the filtrate and the parent untreated extractives solution and after desorption with absolute EtOH. To exclude the coelution of other phytochemicals applying the above mentioned preconcentration procedure, thin layer chromatography (TLC) analyses of the filtrate and the desorbed solutions were carried out coupled with the HPLC analyses. Further assays for the same purpose consisted in the total polyphenol content determination that was accomplished using the same general procedure (e.g. Folin Ciocalteau method) as described in the literature.⁶

For the majority of compounds under investigation, Mg-based clays has shown a strong preference for the adsorption and / or inclusion of natural compounds incorporating a polyphenolic portion, pending the presence of a lamellar structure. This hypothesis may explain why both MgO and Mg(OH)₂ are not effective like other Mg-containing material as they are not featured by such a structural arrangement. The above-mentioned method resulted to be highly reproducible for a wide panel of bioactive principles, without interfering with their stability to oxidation, temperature, pH and light exposure. In fact, further experiments carried out in this perspective aim to demonstrate how their effectiveness is not affected by extreme conditions, providing the release of the bioactive compound intercalated and preserving its chemical structure.

Moreover, we found out that in some cases, these latter have demonstrated to increase the active compound's effects respect to the parent preparation. In this perspective, the final samples obtained in the process resulted even more effective than the crude ones, hence it could be useful in food additives industry or in pharmacological treatments. This was deeply demonstrated for example in the concentrated pomegranate juice, for which the methodology reported has shown to improve its antioxidant and radical scavenging activity thanks to an enrichment in the polyphenol fraction, difficult to recover from the parent matrix because of the fast decomposition of the thermolabile phytochemicals. The Trolox equivalent antioxidant capacity (TEAC)/ABTS assay were used to confirm these results, underlining the versatility of the solid supports herein under study.

Besides, in liquor industry is often necessary the use of flavor and odor correctors obtained from the same vegetal parent extracts to enhance the taste of the final product in order to increase the compliance of the consumers. To this concern it must be reminded how the phytochemical and overall organoleptic qualities and properties of the vegetal extracts strictly depend on parameters related to the plant like their age, the period of collection, climate, geographical factors, storage, overall processing, the intimate method by which the extraction is carried out, and similar.⁷ Due to foregone variations in these factors, entire batches of the liquor production must be very often discarded due to malevolent odors and flavors ("herbaceous like" or "earthy-like") mainly because of a low content of the bitter principles and to the presence of excesses of lignin depolymerization derived compounds, like diterpenes, xanthones, or volatiles.

Therefore, the use of solid-phase extraction processes performed with LDHs, could be a satisfying meaning for the purpose, allowing to obtain pure naturally derived food additives in very good yields from alternative natural plant sources, which are not protected by law like Gentiana lutea, or other Gentianaceae (e.g. those encoded by Chinese, Tibetan, Indian, and Ayurvedic medicine) and Swertia spp, for whose harvest a particular permission from local government authorities is required.

In this perspective, the present Doctorate Course, is carried out through a collaboration with a liquor industry settled in Abruzzo region. Its purpose is to assess the reproducibility of the above-mentioned methods in an industrial scale and to verify the parameters obtained in terms of phytochemical fingerprints of the final products. Since the need for optimized outputs is reflected even in the selection of the best extraction method depending on the parent vegetal matrix, our contribution to this aim was to screen the best methodology among the well-known ones and to promote the new procedure set up.

Seemingly, the biocompatibility and non-toxicity of these materials for patients make them suitable for the intercalation of bioactive compounds as potential drug delivery and vehiculation systems, and in this context further studies will be carried out in the next future.

References
1. Idolo M., Motti R., Mazzoleni S. Journal of Ethnopharmacology. 2010. DOI: 10.1016/j.jep.2009.10.027
2. Liling W., Yifeng Z., Yanbin W., Yuchuan Q., Bentong L., Minge B. Food and Bioproducts Processing. 2019. DOI: 10.3892/mmr.2019.10625
3. Kaipper B. I. A., Madureira L. A. S., Corseuil H. J. Braz. Chem. Soc. 2001. DOI: 10.1016/j.microc.2004.02.014
4. Buszewski, B., Szultka, M. Reviews in Analytical Chemistry. 2012, 198-213. DOI: 10.1080/07373937.2011.645413
5. Choy J.H., Choi S. J., Oh J. M., Park T. Applied Clay Science. 2007, 122-132. DOI: 10.1016/j.clay.2006.07.007
6. Pavun L., Durdevic P., Jelikic-Stankov M., Dikanovic D., Uskokovic-Markovic S. Czech journal of food sciences. 2018, 36, 233-238. DOI: 10.17221/211/2017-CJFS
7. Markovic T., Radanovic D., Nastasijevic B., Antic-Mladenovic S., Vasic V., Matkovic A. Industrial Crops and Products. 2019, 132, 236-244. DOI: 10.1016/j.indcrop.2019.02.027

Current bioanalytical methods for the quantification of peptides and proteins used in biomedical research and pharmaceutical R&D are predominantly based on immunoassay methodologies, such as enzyme-linked immunosorbent assays (ELISA). Although these antibodybased techniques are exceptionally sensitive, they suffer from issues, such as cross-reactivity, long development time, and high variability. Recently, liquid chromatography-mass spectrometry (LC-MS)-based quantification techniques for large molecules, including the bottom-up and top-down approaches, are gaining popularity due to their shorter development time, higher specificity, and ability to detect degradation products and post-translational modifications (PTMs). The multiple reaction monitoring (MRM)-based bottom-up LC-MS/MS approach, which targets enzymatic digestion-produced signature peptides as surrogates in a quantification assay, is a sophisticated method with good sensitivity, selectivity, and multiplex capacity, although it may miss critical information such as PTMs, sequence variants and isoforms, and truncation and degradation products.

While tandem quadrupole MS is the most widely adopted platform for quantification, use of high-resolution mass spectrometers (HRMS), with their ability to provide high selectivity and collect both quantitative and qualitative data, is steadily increasing.

A few advantages of HRMS for large molecule bioanalysis include high mass resolution detection of peptide fragments, for challenging samples, and when interference from endogenous species is a problem. Quantification of large proteins (>15 kDa) at the intact level, using the extended mass range, is something that cannot be done with current triple quadrupole mass spectrometers. Adding to this, there are simply some questions only HRMS can answer, and these are typical of a qualitative nature, for example; large molecule metabolite identification and biopharmaceutical characterization.

HRMS adds flexibility to answer questions of both a quantitative and qualitative nature. Scientists in DMPK groups, not only want to quantify the clearance of a drug, but also understand how it is metabolized. The qualitative capability of HRMS enables analysts to identify and track multiple metabolites without prior knowledge, and this is particularly important when, for example, a biotherapeutic contains non-natural amino acids.

Example of both LC MSMS and HRMS application will be presented.

In the preclinical phase of the process that lead to the approval of a new drug, an important role is played by the drug disposition studies, that are part of the ADME studies, that allow to characterize the fate of a test item administered in vivo. In particular, the regulatory authorities require that ADME studies be performed with radiolabeled material, to better understand the absorption, distribution, metabolism and excretion process.

The most important advantages of using labelled material instead of unlabeled material is that radioactivity could be easily detected in every matrix and allow the quantification of all the radioactive drug related material that forms, after administration in vivo, due to the metabolic enzymatic activity. For this reason, the use of radioactivity to investigate the ADME process allow the detection not only of the parent compound, but also of the known and unknown in vivo metabolites for a better characterization of the drug in object.

An overview of the most used radio isotopes, whit their main characteristics, and the relevant aspects for the conduct of drug disposition studies with radiolabeled drugs for ADME profiling will be presented. The pros and cons of using radioactivity to perform ADME studies will be exposed.

The general experimental design for conducting studies of pharmacokinetic on blood and plasma, quantitative whole body autoradiography (QWBA) and mass balance will be also described for characterization, respectively, of absorption, distribution, excretion and metabolism process in animal models.

Particular attention will be put in the description of quantitative whole body autoradiography (QWBA), an imaging technique to investigate the distribution in the whole animal body: the method will be presented with details on the experimental procedure to obtain animal sections, the digital image acquisition system and the analytical software to obtain quantitative distribution results. Different examples of tissue distribution pattern will be presented to better understand the potential of the technique.

Autoradioluminogram of a rat orally dosed with a 14C radiolabeled test item

Oligonucleotide drugs have increasingly gained attention and the disease areas for which these therapeutic agents are evaluated have been rapidly expanding. In order to determine the PK/PD properties of oligonucleotides, sensitive and selective quantitative bioanalytical methods are required. LC-MS has emerged as a powerful tool and one of the most selective bioanalytical technologies to quantify a variety of oligonucleotide therapeutics in biological matrices. Compared with alternative assay types (e.g. hybridization-ELISA), LC-MS methods can selectively quantify the full-length oligonucleotides and their major metabolites, have a much wider dynamic range and do not require any specific reagents. However, significant challenges should be addressed while developing an LC-MS assay for oligonucleotides, including their very high plasma protein binding, non specific binding to containers, lack of chromatographic retention, multiple charge states and formation of cation adducts affecting the LC-MS method performance. The sensitivity of LC-MS methods for quantification of oligonucleotides has significantly improved since the last few years and reaching sub ng/mL lower limits of quantification is now feasible. This is mainly due to improved MS equipment, consolidated extraction techniques and the adoption of state of the art chromatographic technologies. The LC-MS sensitivity is usually sufficient to support preclinical studies, particularly for analysis of tissues where these drugs exhibit their efficacy and are present at high concentrations. However, the sensitivity might not be sufficient to support clinical studies where the administered doses and the observed exposures are expected to be lower. Sensitivity will be also challenging in certain matrices, e.g. in plasma or serum when the administration of the oligonucleotide drug is not systemic.

In all cases defined above, switching technology (e.g. LC-MS to hELISA) in order to meet a sensitivity target should be carefully evaluated because the selectivity of these assays can be different and a direct comparison of the obtained exposures may not be possible.

According to the MIST guidance, the metabolites of oligonucleotide drugs should be quantified in case these significantly contribute to the pharmacological and toxicological effect. Again, the use of LC-MS offers significant advantages compared to other technologies due to the increased selectivity. However, additional complexity is due to the fact that the metabolic profiles of ASOs in plasma and in tissues are typically very different, resulting in different sensitivity requirements.

Considering all the above considerations and challenges, a proposed strategy is to generate preliminary metabolite i.d. information in plasma and tissues before the quantitative technology is selected and bioanalytical methods are fully validated to support regulatory toxicological studies. The pharmacological activities of predictable shortmers should also be evaluated in order to determine what analytes require quantification. The selectivity of LC-MS and hELISA assays vs. shortmers should also be assessed in order to select the technology to fully validate for quantification.

All factors above will be discussed in this presentation, which includes an overview of recent advances in LC-MS bioanalysis of oligonucleotides, along with examples showing how different experimental conditions will affect the performance of the assay. Case studies will also be presented showing how the preliminary metabolite information and a comparison of selectivity/sensitivity of different technologies will help the bioanalytical team implement a proper analytical strategy in order to avoid complex bridging studies at later stages of drug development.

The raise of novel therapeutic modalities and the discovery and improvement of novel biomarker has changed the state of the art of the bioanalytical arena leading to the development and maturation of a diverse suite of technologies.

The requirement for greater sensitivity, selectivity, speed, and cost efficiency is driving the development of new tools such as ultrasensitive and multiplex equipment.

As bioanalytical technologies improve, the demand for large sample volumes for the quantitative bioanalysis of new chemical and biological entities as well as endogenous biomarkers in liquid biological matrices has significantly reduced.

There has been an increasing interest in the development and implementation of microsampling techniques enabling the collection of limited volume of biosamples (less than 50 µL).

This has massive implications and benefits for both nonclinical and clinical studies like reducing animal use in toxicology studies (according to the principle of 3Rs) or improving patient care allowing the self-collection of samples to enable decentrilised clinical trial.

The need of patient centric sampling techniques compared to conventional procedures has been of highlighted especially during the COVID 19 pandemic period where the demand for home sampling technologies has boosted their adaptation.

The bioanalysis of microsamples brings a new set of responsibilities and challenges like changes to the standard process of handling the biosamples as well as the need for additional experiments compared to those normally performed for samples collected by standard sampling procedures.

The guidelines providing recommendations for the validation of bioanalytical methods are evolving including the use of alternative microsampling collection tools.

Although these technologies are starting to be used more frequently, the full potential have not yet been explored. The awareness of their benefits and the common effort that will be made by all the stakeholders in the drug development process towards a human centric health care approach will be detrimental to overcome the actual challenges for their implementation.

Bioanalytical (BA) Labs in pharmaceutical companies are actually responsible for the production of a massive amount of samples analysis.

Main work of the BA Labs is the quantitation of the drugs and metabolites in the plasma samples arising from many different types of pre-clinical (GLP) and clinical studies (GCP). The plasma drug concentration data obtained after the samples analysis are then used for pharmacokinetic and toxicokinetic evaluations for the New Chemical Entities (NCE) before market authorization. The purpose and the usage of this data leads to consider these as ”extremely” critical data.

A strong and robust computerized system, a Laboratory Information Management System (LIMS) that follows data from the production to the report generation and to the data audit is therefore absolutely necessary and requested.

A LIMS is the key for the Analysts to comply with the GxP requests following the last Data Integrity Guidelines during all the Sample and Result Life cycles.

A general overview of results data production and of management of the hidden data required to support the results will be presented.

A strong and robust computerized system, a Laboratory Information Management System (LIMS) that follows data from the production to the report generation and to the data audit is therefore absolutely necessary and requested.

A LIMS is the key for the Analysts to comply with the GxP requests following the last Data Integrity Guidelines during all the Sample and Result Life cycles.

A general overview of results data production and of management of the hidden data required to support the results will be presented.

Patient safety is paramount in the drug development phase therefore the Regulatory Authorities established criteria for the performance of the non-clinical safety studies with the purpose to guarantee the quality of data and for clinical studies with the purpose to protect the rights, safety and well-being of trial subjects. These criteria are defined in the Good Laboratories Practices (GLP) developed by OECD and are applicable to non-clinical health and environmental safety studies and the Good Clinical Practices (GCP) developed by ICH applicable to Clinical trials.

The aim of this presentation is to provide an overview of GLP and GCP quality systems and to introduce the critical elements of GLP quality system focusing on requirements and related challenges specific for analytical laboratories and on the elements of GCP quality system (GCLP) that apply to laboratories that perform analysis of clinical samples.

In this lesson an overview of LC-MS/MS method applied to Antibody Drug Conjugates (ADC) Bioanalysis will be presented.

After an introduction on ADC mode of action, the main assays needed for the ADC pharmacokinetic profile set up will be discussed. In addition, an LC-MS ligand binding hybrid method, based on high-resolution mass spectrometry, to detect intact protein and determine DAR values will be introduced.

Different free toxin assays will be described reporting both protein precipitation technique and solid phase sample preparation techniques.

The Ab-conjugated payload assay will be covered by considering an enzymatic dependent toxin cleavages. This method consists of an immunocapture step that selectively binds IgG antibodies. Subsequently an enzymatic cleavage step was applied to the immobilized ADC on the plate to generate free payload. Eluates form the plate were subjected to a protein precipitation technique (PPT) step and supernatants are finally analyzed by LC-MS/MS to quantify the Ab-conjugated payload.

Data related to qualification of the method will be presented and a comparison of the results obtained using antibody conjugated payload and conjugated antibody methods will be described as well as the application to ADC stability studies.

In addition, a novel Affinity Capture LC-MS (AFC LC-MS) method that characterizes ADC molecules irrespective of their site of conjugation and linker (cleavable or non-cleavable) type will be presented. This method is particularly useful for ADCs carrying plasma unstable cytotoxic small-molecule drugs, because, in these cases, conjugated payload and free payload LC-MS methods require derivatization of the reactive group on the small-molecule drug.

AFC LC-MS involves immobilization of a biotinylated capturing reagent (e.g., target antigen, anti-idiotype Ab etc.) on streptavidin-coated beads. The beads are then washed multiple times with buffers and/or organic solvents to remove non-specific binders. For ADCs that carry glycans molecule on the CH2 domain of antibody, an additional deglycosylation step may be needed. An elution step at acidic or basic pH is finally required to detach molecule of interest before injecting onto LC-MS system.

High/Ultra Performance Liquid Chromatography (HPLC/UPHLC), often combined with mass spectrometry, has become an increasingly applied methodology in the field of molecular characterization and quantification. In the pharmaceutical field it is used since the beginning, in the development and characterization phase of new compounds, till to the clinical evaluation phases (pharmacokinetic and toxicity analysis). Sample preparation is a process that aims to the selective isolation of the target analyte from the matrix. Efficiency of sample preparation is determinant for a good chromatographic analysis, especially with biological samples (from animals or humans). It could take the 80% of the total time of the bioanalysis and it is the step with highest risk of errors.

The purpose of this presentation is to provide an overview of the main biological sample preparation techniques by providing examples of their practical application in different pharmacological fields.

Sample preparation techniques
Blood and plasma are the most analyzed biological matrix for their easy way of collection. Among the preparation techniques we have some easy and fast methods such as protein precipitation (PP) or liquid-liquid extraction (LLE), or techniques that require the development of more complex methods such as solid phase extraction (SPE) and immunoprecipitation. The choice of technique to use varies based on various factors, including laboratory requirements, sample volume, chromatographic instrument availability, and chemical characteristics of the molecule.

Biological matrix
Plasma is the most used biological matrix, but working in the pharmacological field often happens to have to analyze other biological matrix even complex ones for different types of studies. Among the other matrix analyzed there are urine (especially toxicology studies), cells, tissues and cerebrospinal fluid. The evaluation of the type of matrix is important for the determination of the analytical preparation technique to be used.

Dried sample spot device
The use of dried blood spots (DBS), blood droplets deposited and dried on filter paper, is a long-established technique for the screening of errors in the metabolism of newborns. Since this is a dispersion on paper, the volume of deposited matrix is limited, so up to a few years ago the technique was not very usable for the development of pharmaceutical products. However, thanks to the great technical improvement of the analysis tools, especially in the terms of sensitivity, DBS and dried plasma spots are increasingly used, by virtue of their advantages compared to the classic sampling in the test tube. The advantages are above all the reduction of the shipping / storage cost of the sample, reduction of the sampling volume and the elimination of the infectious risk of the sample itself.

Introduction
Cannabis sativa L. is an herbaceous plant belonging to the Cannabaceae family. Nowadays there is plenty of literature on this plant, which is well-known for both its bioactivity and social implications [1]. Non-psychoactive or fibre-type C. sativa, commonly known as hemp, differs from medicinal and recreational chemotypes for its cannabinoids profile. Indeed, hemp is a rich source of non-psychoactive cannabinoids, contrary to medicinal and recreational Cannabis varieties, which are characterized by a remarkable high content of the psychoactive compound Δ⁹-tetrahydrocannabinol (Δ⁹-THC), together with its acidic precursor Δ⁹-tetrahydrocannabinolic acid (Δ⁹-THCA) [1-2]. In general, the main cannabinoids present in hemp inflorescences are cannabidiolic acid (CBDA) and its decarboxylated form, namely cannabidiol (CBD), but other cannabinoids can be also found, including cannabigerolic acid (CBGA), cannabigerol (CBG), cannabichromenic acid (CBCA) and cannabichromene (CBC) [2]. Scientific studies devoted to unraveling the biological potential of hemp and non-psychoactive cannabinoids have recently increased in number [3-6]. The growing interest in both the pharmacological activities and toxicity of cannabinoids together with a still unclear legislation regarding the use of non-psychoactive C. sativa for pharmaceutical and nutraceutical purposes, triggered the race at the development of highly efficient and reliable analytical methods for its characterization in many different matrices [7]. The most frequently analyzed sample is represented by hemp raw plant material, and in particular its inflorescences, to have a full profile of the plant metabolome and to monitor the content of bioactive compounds in the perspective of a suitable standardization of derived extracts [8-10]. Among analytical techniques, high-performance liquid chromatography (HPLC) coupled with diode-array detection (UV/DAD) and/or mass spectrometry detection (MS) is the most exploited one for the analysis of cannabinoids [9-11].
In addition to cannabinoids, terpenes, which are the constituent of hemp essential oil, contribute to the aroma of various hemp genotypes, and so far, around 140 different terpenes have been reported in hemp. The main compounds belong to the class of monoterpenes (i.e., a-pinene and β-myrcene) and sesquiterpenes (β-caryophyllene) [3,12]. In this case, the technique of choice for the quali - and quantitative analysis is gas chromatography (GC), coupled with a flame ionization detector (FID) and/or mass spectrometry (MS). C. sativa terpenes have been less studied than the cannabinoids, and the potential interaction between these two classes of compounds has barely been studied at all. The hypothesized interactions between various cannabinoids and terpenes to produce unique outcomes is known as the entourage effect [13]. If terpenes modulate cannabinoids, then it might be possible to identify terpenes that maximize the therapeutic efficacy of cannabinoids while reducing unwanted side effects.
In the light of all the above, this PhD project was focused on the development of new methods for the preparation and chemical characterization of non-psychoactive C. sativa extracts and on the evaluation of their bioactivity both in vitro and in vivo. In particular, the first part of the PhD program was aimed at the assessment of the antiproliferative activity of hemp extracts on different cancer cell lines and at the investigation of the mechanism of cell death mediated by both the extracts and pure compounds. In the second part of the project, olive oil extracts from a non-psychoactive C. sativa variety having a high content of CBD were prepared and fully characterized to be tested against chronic pathologies of the central nervous system (CNS), focusing on in vivo models of neuropathic pain and epilepsy. In this case, the role of the volatile components and the synergism with cannabinoids was investigated. Finally, an analytical section was developed with a new and highly efficient HPLC method optimized here for the first time for the simultaneous separation of 14 cannabinoids.

Extraction and chemical characterization of non-psychoactive C. sativa extracts for in vitro studies
This first project was focused on the investigation of the possible role of non-psychoactive C. sativa extracts as antiproliferative agents. For the in vitro assays, raw and decarboxylated ethanolic extracts were prepared starting from three fibre-type C. sativa varieties having a different phytochemical composition, a CBD-type, a CBG-type and a hybrid-type.
First, it was necessary to fully characterize the extracts by means of ultra high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-HRMS). To do this, an inclusion list of more than 200 compounds based on the chemical structures previously described in the literature was built and a target metabolomic analysis was performed in this way. This technique allowed us to identify minor compounds present in the extracts and to have a complete profiling of cannabinoids. Then, HPLC-UV was used to quantify the major cannabinoids in the extracts.
Cell lines of different embryological origin were then selected for the cell viability assays: the K562 human chronic myelogenous leukemia cell line, the HT29 human colorectal adenocarcinoma cell line and the U87MG human glioblastoma cell line. The decarboxylated CBD-type extract was the most active one in inhibiting cell proliferation and the K562 cell line was the most responsive to this treatment, with an IC₅₀ value of 11 µg/mL. In addition, the extract showed a lower IC50 value compared to pure CBD. K562 cells were used for the detailed investigation of the mechanism of cell death mediated by the CBD-rich extract. The cytofluorimetric analysis of K562 treated cells revealed that the antiproliferative effect was due to the induction of apoptosis, as shown by an increase of cell population annexin V positive. The expression and activity of the main apoptosis-related proteins was investigated through western blot, and it was proved that the mechanism of cell death did not involve the main pro- and anti-apoptotic markers, such as p53, Bcl-2 and Bcl-xl. The extract was able to increase the cytoplasmic levels of cytochrome c. Moreover, caspase 3 and 7 seemed to be involved in the mechanism of apoptosis.
To conclude, CBD can be involved in the antiproliferative activity of the CBD-rich extract even if the role of other minor compounds cannot be excluded now. The mechanism of action is still under investigation using both in silico and proteomic analysis, in collaboration with Prof. Maria Chiara Monti of the University of Salerno.

Extraction and chemical characterization of non-psychoactive C. sativa extracts for in vivo studies
This second project was focused on the investigation of the possible role of non-psychoactive C. sativa extracts against CNS chronic diseases. For the in vivo assays, the choice of the solvent used for the extraction was crucial since it needed to be suitable for oral administration by gavage. Olive oil was chosen as the extraction solvent and non-psychoactive C. sativa olive oil extracts were prepared and tested in animal models of peripheral neuropathy and epilepsy. Two olive oil extracts were prepared starting from a decarboxylated CBD-type hemp variety, including one extract rich in cannabinoids (K1) and one having a high content of both cannabinoids and terpenes (K2). Concerning the chemical characterization of the above-mentioned extracts, HPLC-HRMS and HPLC-UV were used for the qualitative and quantitative analysis of cannabinoids. The volatile component of the extracts was fully characterized and quantitated by means of a new method based on GC-MS. Terpenes were identified according to their MS spectra, based on the full-scan GC-MS data, and the most representative compounds were quantified in the selected ion monitoring (SIM) mode, using a method developed for the first time in this work. Concerning the in vivo model of neuropathy, the anti-hyperalgesic effect of the oils after oral administration was assessed by measuring mechanical and thermal allodynia in the spare nerve injury (SNI) model, in collaboration with Prof. Nicoletta Galeotti of the University of Florence. The mechanism of action was investigated using the spinal cords samples. Oral administration of the extract K2 at 25 mg kg-1 ameliorated mechanical and thermal allodynia in SNI mice, with a rapid and a long-lasting effect, without inducing an alteration of locomotor activity, with an efficacy higher than K1. The extract reduced p-ERK, p-p38 and p-JNK1 protein levels compared to the untreated mice and downregulated protein expression of neuroinflammatory factors (HDAC-1, p-65, NOS2). These effects were reverted by the administration of a CB2 antagonist (AM630), but not by the CB1 antagonist (AM251), suggesting microglial CB2 modulation as a key regulator for the analgesic effect. These results suggested that oral K2 administration attenuated SNI-induced neuropathic pain symptoms by reducing spinal neuroinflammation. These data strongly underline the key role of the synergism between cannabinoids and terpenes for the management of neuropathy related symptoms.
Moving to the in vivo model of epilepsy, the same extracts K1 and K2 were tested in the 6-Hz corneal stimulation murine model to evaluate the antiepileptic activity of the oils compared to pure CBD [14]. This was conducted in collaboration with Prof. Giuseppe Biagini of the University of Modena and Reggio Emilia. Hemp oils were administered 1 h before the 6-Hz corneal stimulation test (44 mA). Mice were stimulated once a day for 5 days and evaluated by video-electrocorticographic recordings and behavioral analysis. Neuronal activation was assessed by FosB/?FosB immunoreactivity. Both oils significantly reduced the percentage of mice experiencing convulsive seizures compared to control but only the oil enriched with terpenes significantly accelerated full recovery from the seizure. The crucial role of the volatile component of K2 in the overall activity of the extract, together with the presence of CBD, could be supported by the fact that CBD alone, at a low dose, seemed not to be effective. The results obtained on the 6-Hz corneal stimulation model suggested a role of terpenic components in enhancing the antiepileptic activity of cannabinoids in K2 oil, compared to K1 and pure CBD, even if other minor compounds in the extracts can contribute to the overall activity. To deeper investigate the possible synergism of CBD and β-caryophyllene (BCP), which are the two main compounds among cannabinoids and terpenes, respectively, an in silico structure-based approach was performed on CB2 receptors, where BCP acts as a full agonist, while CBD seems to be a partial agonist [15,16]. This study was performed in collaboration with Prof. Fabrizio Manetti of the University of Siena. CBD and BCP were simultaneously docked into the binding site of CB2 (PDBID: 5zty), using AutoDock Vina 1.2.0. CBD was predicted to bind close to the binding site entrance, while BCP was docked deeper into the binding site. Together, CBD and BCP were able to resemble the experimental binding mode of the reference ligand and the interactions with the binding site residues better than CBD or BCP alone, which could explain the higher activity of K2 compared to K1. In vitro binding and functional assays are currently ongoing to validate the in silico results.

Development of a new HPLC method for the separation of cannabinoids using DoE
As for the analytical section of this project, the work was addressed at the development of an innovative HPLC method for the simultaneous separation of cannabinoids using the Design of Experiments (DoE), in collaboration with Dr. Caterina Durante of the University of Modena and Reggio Emilia. The aim of the work was to develop a simple, rapid and reliable reverse-phase HPLC method for the separation of 14 cannabinoids, which were carefully selected in order to represent the main compounds found in C. sativa.
A face-centered central composite design (FCCD) was applied for the design of the experimental tests, systematically considering the different chromatographic parameters affecting resolution and their range of variability. To study how the investigated variables affected the resolution of the chromatographic peaks, a major component analysis (PCA) was carried out. Then, a multilinear regression was used to develop a mathematical model capable of identifying the most influential parameters. The developed method allowed the separation of 13 out of 14 cannabinoids. Since the separation of CBG and cannabinerol (CBNe), which are geometric isomers, was not obtained under the applied conditions, HPLC coupled with a triple-quadrupole mass analyzer was used, monitoring the specific transitions of each compound in the multiple reaction monitoring (MRM) mode. In this way it was possible to discriminate between the co-eluted compounds. Finally, the method was applied to C. sativa extracts having a different cannabinoid profile from recreational-type, drug-type and fibre-type varieties to demonstrate the real applicability of the optimized conditions.

Conclusions
As an overall conclusion, during this PhD project the potential of C. sativa as a source of bioactive compounds was assessed, with particular attention to development of efficient analytical methods for the chemical characterization of extracts. A CBD-rich extract showed antiproliferative activity in an in vitro model of chronic myelogenous leukemia and the results revealed an induction of the apoptotic process. Even if further work is necessary to elucidate the mechanism/s of action involved in the activity of this hemp extract, the research strongly supported the CBD-rich extract and, therefore, its main component CBD as a possible candidate for future therapy of myeloproliferative diseases either alone or in association with other anticancer drugs. Concerning the in vivo studies, the results suggested that cannabinoids and terpenes may act synergistically to enhance the bioactivity of the phytocomplex against neuropathic pain and epilepsy. Future work in this ambit should be focused on the assessment of the bioactivity of K2 oil, as well as its main compounds such as CBD and β-caryophyllene, using additional in vivo models.
The crucial role of HPLC and GC methods, specifically developed in this study for a complete and reliable analysis of cannabinoids and terpenes, highlights the importance of pharmaceutical analysis to ensure the quality of the samples to be used in the biological assays as well as the reproducibility of the results, in the concrete perspective of a translational clinical field.

References
[1] Aliferis K.A., Bernard-Perron D. Front. Plant. Sci. 2020, 11: 554.
[2] Bonini S.A., Premoli M., Tambaro S., Kumar A., Maccarinelli G., Memo M., Mastinu A. J. Ethnopharmacol. 2018, 227: 300-315.
[3] Iseppi R., Brighenti V., Licata M., Lambertini A., Sabia C., Messi P., Pellati F., Benvenuti S. Molecules, 2019, 24: 2302.
[4] Pellati F., Borgonetti V., Brighenti V., Biagi M., Benvenuti S., Corsi L. Biomed. Res. Int. 2018, 2018: 1691428.
[5] Atalay S., Jarocka-Karpowicz I., Skrzydlewskas E. Antioxidants. 2020, 9: 21.
[6] Anceschi L., Codeluppi A., Brighenti V., Tassinari R., Taglioli V., Marchetti L., Roncati L., Alessandrini A., Corsi L., Pellati F. Phytotherapy Res. 2022, 36: 914-927.
[7] Brighenti V., Protti M., Anceschi L., Zanardi C., Mercolini L., Pellati F. J. Pharm. Biomed. Analysis. 2021, 192: 113633.
[8] Citti C., Russo F., Sgro S., Gallo A., Zanotto A., Forni F., Vandelli M.A., Lagana A., Montone C.M., Gigli G., Cannazza G. Anal. Bioanal. Chem. 2020, 412: 4009-4022.
[9] Ternelli M., Brighenti V., Anceschi L., Poto M., Bertelli D., Licata M., Pellati F. J. Pharma. Biomed. Analysis. 2020, 186: 113296.
[10] Brighenti V., Marchetti L., Anceschi L., Protti M., Verri P., Pollastro F., Mercolini L., Bertelli D., Zanardi C., Pellati F. J. Pharma. Biomed. Analysis. 2021, 206: 114346.
[11] Protti M., Brighenti V., Battaglia M.R., Anceschi L., Pellati F., Mercolini L. ACS Med. Chem. Lett. 2019, 10: 539-544.
[12] Fiorini D., Scortichini S., Bonacucina G., Greco N.G., Mazzara E., Petrelli R., Torresi J., Maggi F., Crespi M. Ind. Crops. Prod. 2020, 154: 112688.
[13] Anand U., Pacchetti B., Anand P., Sodergren M.H. Pain Manag. 2021, 11: 395-403.
[14] Costa A.M., Senn L., Anceschi L., Brighenti V., Pellati F., Biagini G. Pharmaceuticals. 2021, 14: 1259.
[15] Gertsch J., Leonti M., Raduner F., Zimmer A. PNAS. 2008, 105: 9099-9104.
[16] Tham M., Ylmaz O., Alaverdashvili M., Kelly M., Denovan-Wright E.M., Laprairie R.B. Br. J. Pharmacol. 2019, 176: 1455-146