A Look into Ocular Diseases: The Pivotal Role of Omics Sciences in Ophthalmology Research.

Precision medicine is a new medical approach which considers both population characteristics and individual variability to provide customized healthcare. The transition from traditional reactive medicine to personalized medicine is based on a biomarker-driven process and a deep knowledge of biological mechanisms according to which the development of diseases occurs. In this context, the advancements in high-throughput omics technologies represent a unique opportunity to discover novel biomarkers and to provide an unbiased picture of the biological system. One of the medical fields in which omics science has started to be recently applied is that of ophthalmology. Ocular diseases are very common, and some of them could be highly disabling, thus leading to vision loss and blindness. The pathogenic mechanism of most ocular diseases may be dependent on various genetic and environmental factors, whose effect has not been yet completely understood. In this context, large-scale omics approaches are fundamental to have a comprehensive evaluation of the whole system and represent an essential tool for the development of novel therapies. This Review summarizes the recent advancements in omics science applied to ophthalmology in the last ten years, in particular by focusing on proteomics, metabolomics and lipidomics applications from an analytical perspective. The role of high-efficiency separation techniques coupled to (high-resolution) mass spectrometry ((HR)MS) is also discussed, as well as the impact of sampling, sample preparation and data analysis as integrating parts of the analytical workflow.

The influence of drying and storage conditions on the volatilome and cannabinoid content of Cannabis sativa L. inflorescences.

The increasing interest in hemp and cannabis poses new questions about the influence of drying and storage conditions on the overall aroma and cannabinoids profile of these products. Cannabis inflorescences are subjected to drying shortly after harvest and then to storage in different containers. These steps may cause a process of rapid deterioration with consequent changes in precious secondary metabolite content, negatively impacting on the product quality and potency. In this context, in this work, the investigation of the effects of freeze vs tray drying and three storage conditions on the preservation of cannabis compounds has been performed. A multi-trait approach, combining both solid-phase microextraction (SPME) two-dimensional gas chromatography coupled to mass spectrometry (SPME-GC × GC-MS) and high-performance liquid chromatography (HPLC), is presented for the first time. This approach has permitted to obtain the detailed characterisation of the whole cannabis matrix in terms of volatile compounds and cannabinoids. Moreover, multivariate statistical analyses were performed on the obtained data, helping to show that freeze drying conditions is useful to preserve cannabinoid content, preventing decarboxylation of acid cannabinoids, but leads to a loss of volatile compounds which are responsible for the cannabis aroma. Furthermore, among storage conditions, storage in glass bottle seems more beneficial for the retention of the initial VOC profile compared to open to air dry tray and closed high-density polyethylene box. However, the glass bottle storage condition causes formation of neutral cannabinoids at the expenses of the highly priced acid forms. This work will contribute to help define optimal storage conditions useful to produce highly valuable and high-quality products.

Sustainable cannabinoids purification through twin-column recycling chromatography and green solvents.

In the present study, twin-column recycling chromatography has been employed for the purification of a Cannabis extract by using a green solvent, ethanol, as the mobile phase. In particular, the complete removal of the psychoactive tetrahydrocannabinol (THC) from a Cannabis extract rich in cannabidiol (CBD) was achieved under continuous conditions. The performance of the method, in terms of compound purity, recovery, productivity and solvent consumption, was compared to that of traditional batch operations showing the potential of the twin-column recycling approach. The employment of a theoretical model to predict the band profiles of the two compounds during the recycling process has facilitated method development, thus further contributing to process sustainability by avoiding trial and error attempts or at least decreasing the number of steps significantly.

Electrocatalytic Poly(3,4-ethylenedioxythiophene) for Electrochemical Conversion of 5-Hydroxymethylfurfural.

This study investigates the utilization of the conductive polymer poly(3,4-ethylenedioxythiophene) (PEDOT) as a catalytic material for the 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-mediated oxidation of 5-hydroxymethylfurfural (HMF) to 2,5-furandicarboxylic acid (FDCA). PEDOT films doped with different counterions were electrodeposited on graphite foil. In particular, the mobile anion perchlorate and the polymeric ionomers polystyrenesulfonate, Nafion, and Aquivion were used. The electrocatalytic properties of PEDOT films were evaluated toward the TEMPO redox mediator in the absence and the presence of HMF as a substrate for oxidation reactions. The electrocatalytic HMF oxidation was confirmed to occur at PEDOT electrodes, and it was also found that the chemical nature of PEDOT counterions controls the electrocatalytic conversion of HMF by modulating the kinetics of the electrochemical generation of the oxoammonium cation TEMPO(+). Potentiostatic electrolysis experiments showed that both the reference graphite electrode and PEDOT substrates were able to convert HMF to FDCA with an 80% faradaic efficiency (FE) and a >90% yield (FDCA), but, compared to graphite, the complete conversion of HMF to FDCA required a ca. 30% shorter time when using PEDOT electrodes doped with perchlorate or Aquivion, thanks to their ability to sustain a higher current density in the initial phase of the electrolysis. In addition, while all PEDOT films were chemically stable under the electrochemical conditions herein described, only PEDOT films doped with Aquivion were also mechanically robust and stable against delamination. Thus, the new PEDOT/Aquivion composite may represent the best choice for the implementation of PEDOT-based electrodes in TEMPO-mediated electrocatalytic applications.

Enhancing the purification of crocin-I from saffron through the combination of multicolumn countercurrent chromatography and green solvents.

Crocin-I, a valuable natural compound found in saffron (Crocus sativus L.), is the most abundant among the various crocin structures. Developing a cost-effective and scalable purification process to produce high-purity crocin-I is of great interest for future investigations into its biological properties and its potential applications in the treatment of neurological disorders. However purifying crocin-I through single-column preparative chromatography (batch) poses a yield-purity trade-off due to structural similarities among crocins, meaning that the choice of the collection window sacrifices either yield in benefit of higher purity or vice versa. This study demonstrates how the continuous countercurrent operating mode resolves this dilemma. Herein, a twin-column MCSGP (multicolumn countercurrent solvent gradient purification) process was employed to purify crocin-I. This study involved an environmentally friendly ethanolic extraction of saffron stigma, followed by an investigation into the stability of the crocin-I within the feed under varying storage conditions to ensure a stable feed composition during the purification. Then, the batch purification process was initially designed, optimized, and subsequently followed by the scale-up to the MCSGP process. To ensure a fair comparison, both processes were evaluated under similar conditions (e.g., similar total column volume). The results showed that, at a purity grade of 99.7%, the MCSGP technique demonstrated significant results, namely + 334% increase in recovery + 307% increase in productivity, and - 92% reduction in solvent consumption. To make the purification process even greener, the only organic solvent employed was ethanol, without the addition of any additive. In conclusion, this study presents the MCSGP as a reliable, simple, and economical technique for purifying crocin-I from saffron extract, demonstrating for the first time that it can be effectively applied as a powerful approach for process intensification in the purification of natural products from complex matrices.

Enzymatic Synthesis of New Acetoacetate-Ursodeoxycholic Acid Hybrids as Potential Therapeutic Agents and Useful Synthetic Scaffolds as Well.

Ursodeoxycholic acid (UDCA) and acetoacetate are natural compounds present in the human intestine and blood, respectively. A number of studies highlighted that besides their well-known primary biological roles, both compounds possess the ability to influence a variety of cellular processes involved in the etiology of various diseases. These reasons suggested the potential of acetoacetate-UDCA hybrids as possible therapeutic agents and prompted us to develop a synthetic strategy to selectively derivatize the hydroxyl groups of the bile acid with acetoacetyl moieties. 3α-acetoacetoxy UDCA was obtained (60% isolated yield) via the regioselective transesterification of methyl acetoacetate with UDCA promoted by the Candida antarctica lipase B (CAL-B). 3α,7ß-bis-acetoacetoxy UDCA was obtained instead by thermal condensation of methyl acetoacetate and UDCA (80% isolated yield). This bis-adduct was finally converted to the 7ß-acetoacetoxy UDCA (82% isolated yield) via CAL-B catalyzed regioselective alcoholysis of the ester group on the 3α position. In order to demonstrate the value of the above new hybrids as UDCA-based scaffolds, 3α-acetoacetoxy UDCA was subjected to multicomponent Biginelli reaction with benzaldehyde and urea to obtain the corresponding 4-phenyl-3,4-dihydropyrimidin-2-(1H)-one derivative in 65% isolated yield.

Novel insights into the dependence of adsorption-desorption kinetics on particle geometry in chiral chromatography.

The existence of slow adsorption-desorption kinetics in chiral liquid chromatography is common knowledge. This may significantly contribute to worsening the efficiency and kinetic performance of a chromatographic run, especially when high flow rates are employed. Many attempts and protocols have been proposed to access this term, the so-called c ads , but they are based on different (theoretical) assumptions. As a consequence, no official method is available for the estimation of the adsorption-desorption kinetics term. In this work, a novel approach to access c ads is presented. This procedure combines experimental results obtained with kinetic and thermodynamic measurements. The investigations have been performed on two zwitterionic teicoplanin chiral stationary phases (CSPs) based on 1.9 µ m fully porous and 2.0 µ m superficially porous particles (FPPs and SPPs), using Z-D,L-Methionine as probe molecule. Kinetic studies have been performed through the combination of both stop-flow and dynamic measurements, while adsorption isotherms have been calculated through Inverse Method. This study has confirmed that, on both particle formats, analyte diffusion on the surface of the particle is negligible, meaning that adsorption is localized, and it has been demonstrated that adsorption-desorption kinetics is strongly dependent on particle geometry and, in particular, on the loading of chiral selector. These findings are fundamental not only to unravel novel aspects of the complex enantiorecognition mechanism but also to optimize the employment of CSPs for ultra-fast and preparative applications.

Exploring the application limits of different hold-up time markers in supercritical fluid chromatography.

The study focuses on the application range of nitrous oxide as a hold-up time marker in supercritical fluid chromatography (SFC). This compound has been suggested a decade ago to be used as unretained marker, something that the field of SFC was missing for a long time, since its beneficial properties make it an ideal candidate as hold-up time marker. Determination of the hold-up volume and actual volumetric flow rates have always been problematic in SFC due to the compressibility of carbon dioxide and one part of this is the difficulty of hold-up time measurements. Depending on the mobile phase, different methods have been used to measure the hold-up time with varying results. Nitrous oxide and other molecules have been compared in different conditions, mobile phases and stationary phases. In all cases, nitrous oxide gave the lowest elution times. However, detection was difficult in mobile phases containing 10% or more of organic modifier, because most solvents mask the signal of nitrous oxide. Interestingly, the choice of stationary phase also had a slight effect on detection, while different pressure and temperature settings affected each compound in a different manner.

Dimethyl carbonate as a green alternative to acetonitrile in reversed-phase liquid chromatography. Part II: Purification of a therapeutic peptide.

Preparative liquid chromatography in reversed phase conditions (RPLC) is the most common approach adopted in the downstream processing for the purification of therapeutic peptides at industrial level. Due to the strict requirements on the quality imposed by the Regulatory Agencies, routinary methods based on the use of aqueous buffers and acetonitrile (ACN) as organic modifier are commonly used, where ACN is practically the only available choice for the purification of peptide derivatives. However, ACN is known to suffers of many shortcomings, such as drastic shortage in the market, high costs and, most importantly, it shows unwanted toxicity for human health and environment, which led it among the less environmentally friendly ones. For this reason, the selection of a suitable alternative becomes crucial for the sustainable downstream processing of peptides and biopharmaceuticals in general. In this paper, a promising green solvent, namely dimethyl carbonate (DMC) has been used for the separation of a peptide not only in linear conditions but also for its purification through non-linear overloaded chromatography. The performance of the process has been compared to that achievable with the common method where ACN is used as organic modifier and to that obtained with two additional solvents (namely ethanol and isopropanol), already used as greener alternatives to ACN. This proof-of-concept study showed that, thanks to its higher elution strength, DMC can be considered a green alternative to ACN, since it allows to reduce method duration while reaching good purities and recoveries. Indeed, at a target purity fixed to 98.5 %, DMC led to the best productivity with respect to all the other solvents tested, confirming its suitability as a sustainable alternative to ACN for the purification of complex biopharmaceutical products.

Retention mechanism on phosphodiester stationary phases in hydrophilic interaction liquid chromatography and purely aqueous mobile phase part II: Overloading with limited soluble samples.

The adsorption behaviour of caffeine and theophylline under hydrophilic interaction chromatography and purely aqueous conditions was investigated on four phosphodiester stationary phases. Solute adsorption isotherms were determined by frontal analysis or inverse method. The bi-Langmuir model was found to be the best choice to describe the behaviour of caffeine and theophylline adsorption in purely aqueous conditions, whereas the bi-Moreau model describes the adsorption phenomena in HILIC conditions. The results obtained demonstrate that the interaction of caffeine and theophylline with the stationary phase surface varies depending on the mobile phase composition. Both in pure aqueous mobile phase and in HILIC mode, the heterogeneity of the surface of the studied stationary phases is confirmed. In hydrophilic solutions, the sample molecules interact with the stationary phase only. In hydrophobic conditions, a lateral interaction occurs between caffeine or theophylline molecules, which are poorly soluble in acetonitrile-rich solvents. This confirms that the same compound on the same stationary phase can behave rather differently, depending on the mobile phase composition. Thus, the mobile phase may govern and control the retention mechanism.

Dimethyl carbonate as a green alternative to acetonitrile in reversed-phase liquid chromatography. Part I: Separation of small molecules.

Nowadays, environmental problems are drawing the attention of governments and international organisations, which are therefore encouraging the transition to green industrial processes and approaches. In this context, chemists can help indicate a suitable direction. Beside the efforts focused on greening synthetic approaches, currently also analytical techniques and separations are under observation, especially those employing large volumes of organic solvents, such as reversed-phase liquid chromatography (RPLC). Acetonitrile has always been considered the best performing organic modifier for RPLC applications, due to its chemical features (complete miscibility in water, UV transparency, low viscosity etc); nevertheless, it suffers of severe shortcomings, and most importantly, it does not fully comply with Environmental, Health and Safety (EHS) requirements. For these reasons, alternative greener solvents are being investigated, especially easily available alcohols. In this work, chromatographic performance of the most common solvents used in reversed-phase chromatography, i.e., acetonitrile, ethanol and isopropanol, have been compared to a scarcely used solvent, dimethyl carbonate (DMC). The analytes of interest were two small molecules, caffeine and paracetamol, whose kinetics and retention behaviour obtained with the four solvents have been compared, and all contributions to band broadening have been assessed. Results about kinetic performance are very promising, indicating that a small amount (7 % v/v) of DMC is able to produce the same efficiency as a 2.5-times larger ACN volume (18 % v/v), and larger efficiency than alcohols. This paper reports, for the first time, fundamental studies concerning the mass transfer phenomena when DMC is used as an organic solvent in RPLC, and, together with the companion paper, represents the results of a research whose final aim was to discover whether DMC is suitable for chromatographic applications both in linear and preparative conditions.

(L)-Monomethyl Tyrosine (Mmt): New Synthetic Strategy via Bulky 'Forced-Traceless' Regioselective Pd-Catalyzed C(sp2)-H Activation.

The enormous influence in terms of bioactivity, affinity, and selectivity represented by the replacement of (L)-2,6-dimethyl tyrosine (Dmt) instead of Phenylalanine (Phe) into Nociceptin/orphanin (N/OFQ) neuropeptide analogues has been well documented in the literature. More recently, the non-natural amino acid (L)-2-methyl tyrosine (Mmt), with steric hindrance included between Tyr and Dmt, has been studied because of the modulation of steric effects in opioid peptide chains. Here, we report a new synthetic strategy to obtain Mmt based on the well-known Pd-catalyzed ortho-C(sp2)-H activation approach, because there is a paucity of other synthetic routes in the literature to achieve it. The aim of this work was to force only the mono-ortho-methylation process over the double ortho-methylation one. In this regard, we are pleased to report that the introduction of the dibenzylamine moiety on a Tyr aromatic nucleus is a convenient and traceless solution to achieve such a goal. Interestingly, our method provided the aimed Mmt either as N-Boc or N-Fmoc derivatives ready to be inserted into peptide chains through solid-phase peptide synthesis (SPPS). Importantly, the introduction of Mmt in place of Phe1 in the sequence of N/OFQ(1-13)-NH2 was very well tolerated in terms of pharmacological profile and bioactivity.

Understanding the Transition from High-Selective to High-Efficient Chiral Separations by Changing the Organic Modifier with Zwitterionic-Teicoplanin Chiral Stationary Phase.

The retention behavior of small molecules and N-protected amino acids on a zwitterionic teicoplanin chiral stationary phase (CSP), prepared on superficially porous particles (SPPs) of 2.0 µm particle diameter, has shown that efficiency and enantioselectivity, and so enantioresolution, dramatically change depending on the employed organic modifier. In particular, it was found that while methanol permits the boost of enantioselectivity and resolution of the amino acids, at the cost of efficiency, acetonitrile allows for the ability to reach extraordinary efficiency even at high flow rates (with reduced plate height <2 and up to 300,000 plates/m at the optimum flow rate). To understand these features, an approach based on the investigation of mass transfer through the CSP, the estimation of the binding constants of amino acids on the CSP, and the assessment of compositional properties of the interfacial region between bulk mobile phase and solid surface has been adopted.

Development and validation of a GC × GC-ToFMS method for the quantification of pesticides in environmental waters.

Water is a fundamental resource for living things, which is why its control is necessary. The widespread use of pesticides for agricultural and non-agricultural purposes has resulted in the presence of their residues in surface water and groundwater resources. Their presence in water is regulated through different directives, such as the Groundwater Directive, the Drinking Water Directive, and the Water Framework Directive, modified later several times, setting a maximum concentration of 0.1 µg.L-1 for individual pesticides and their degradation products, and 0.5 µg.L-1 for total pesticide residues present in a sample. There are different kinds of pesticides (e.g., organophosphorus and organochlorine pesticides, triazines, chloroacetamides, triazoles, (thio)carbamates) that have diverse chemical structures. Their determination and monitoring in a single analytical procedure are possible through multiresidue methods. In this study, 53 pesticides belonging to different chemical classes and their metabolites were selected based on their local occurrence and investigated in surface water and groundwater from agricultural areas susceptible to pesticide contamination. The methodology consisted of a classical solid-phase extraction (SPE) for the purification and enrichment of the pesticides, with a subsequent analysis in multidimensional gas chromatography coupled to mass spectrometry (GC×GC-MS). The quantification method was validated according to the Eurachem Guide in terms of linearity, precision, accuracy, limit of detection, and limit of quantification. After validation, the method was applied to 34 real-world water samples, and the results were compared with those obtained by a GC-QMS routine method.

The Effect of Different Storage Conditions on Phytochemical Composition, Shelf-Life, and Bioactive Compounds of Voghiera Garlic PDO.

Voghiera garlic is an Italian white garlic variety which obtained in 2010 the Protected Designation of Origin. It is widely used for culinary purposes or as an ingredient for supplement production due to its phytochemical compositions. The storage conditions seem to be crucial to retain the high quality of garlic bulbs and their by-products, taking into account the high importance of organosulfur and phenolic compounds for the bioactive potency of garlic and its shelf-life. This study aims to examine the effect of storage on the phytochemical composition, biological effects, and shelf-life of Voghiera garlic PDO. In detail, we considered (i) -4 °C (industrial storage) for 3, 6, and 9 months; (ii) +4 °C for 3 months (home conservation), and (iii) -4 °C for 3 months, plus +4 °C for another 3 months. We focused our attention on the organosulfur compounds, total condensed tannins, flavonoids, phenolic compounds, and related antioxidant activity changes during the storage period. To evaluate the bioactive effects, the Voghiera garlic extracts at different storage conditions were administered to a breast cancer cell line, while antioxidant and anti-inflammatory activity was detected using macrophage RAW 264.7 cells. We observed a decrease in sulfur compounds after 6 months which correlated to a decrease in bioactive effects, while the number of antioxidant compounds was stable during the storage period, showing the good effect of refrigerated temperature in maintaining garlic bulb shelf-life.

Enrichment and recovery of oligonucleotide impurities by N-Rich twin-column continuous chromatography.

N-Rich is a twin-column continuous chromatography technology well suited for small-scale isolation and the enrichment of product related impurities. For the first time, N-Rich was used for impurity isolation from a double-stranded RNA (dsRNA) therapeutic synthetic oligonucleotide (ON), produced by solid-phase synthesis. By employing the N-Rich process, where the desired impurities are recycled and selectively enriched, and interfering substances are depleted, it was possible to obtain substantial amounts of high purity marginal impurities with a reproducible, automatized, and productive method. The productivity-purity tradeoff inherent to traditional impurity isolation methods, i.e., analytical chromatography, was effectively alleviated. Using N-Rich, satisfactory purity values and mass recoveries of several low-concentrated impurities could be obtained simultaneously. A performance comparison demonstrated an up to 15-fold increase for purity values and up to 20-fold mass impurity isolation and concentration with the N-Rich technology in comparison to conventional isolation procedures, drastically reducing processing times, manual handling, and waste production.

Therapeutic Potential of Allicin and Aged Garlic Extract in Alzheimer's Disease.

Garlic, Allium sativum, has long been utilized for a number of medicinal purposes around the world, and its medical benefits have been well documented. The health benefits of garlic likely arise from a wide variety of components, possibly working synergistically. Garlic and garlic extracts, especially aged garlic extracts (AGEs), are rich in bioactive compounds, with potent anti-inflammatory, antioxidant and neuroprotective activities. In light of these effects, garlic and its components have been examined in experimental models of Alzheimer's disease (AD), the most common form of dementia without therapy, and a growing health concern in aging societies. With the aim of offering an updated overview, this paper reviews the chemical composition, metabolism and bioavailability of garlic bioactive compounds. In addition, it provides an overview of signaling mechanisms triggered by garlic derivatives, with a focus on allicin and AGE, to improve learning and memory.

Boosting the enantioresolution of zwitterionic-teicoplanin chiral stationary phases by moving to wide-pore core-shell particles.

A novel zwitterionic-teicoplanin chiral stationary phase (CSP), based on superficially porous particles (SPPs) of 2.7 µm particle diameter and 160 Å pore size, has been prepared and evaluated towards the enantioseparation of important classes of compounds, including chiral drugs, pesticides, and N-derivatized amino acids. The comparison with two analogous CSPs prepared on SPPs with 2.7 and 2.0 µm particle diameter and 90 Å pore size has revealed that the use of large-pore particles allows to dramatically improve both the enantioselectivity and the resolution-per-analysis-time, at the point that the column prepared with the new CSP outperformed the one packed with the finest particles. On the novel wide-pore CSP, the separation of fifteen racemates of pratical importance was significantly improved in terms of both enantioselectivity and resolution-per-analysis time-compared to the CSPs based on SPPs with smaller pores (90 Å). Such a CSP would be suitable for very fast enantioseparations allowing the saving of solvent for greener high-efficiency/high-throughput applications.

Investigation of retention behavior of natural cannabinoids on differently substituted polysaccharide-based chiral stationary phases under reversed-phase liquid chromatographic conditions.

The growing popularity of cannabis products and recent legalization of cannabis for recreational purposes have contributed to the increase of the demand for analytical methods able to give a detailed characterization of cannabis samples and derivatives. In this context, one of the aspects that is strongly emerging is about the hazardous potential of uncharacterised minor cannabinoids, including chiral ones, for which achiral potency testing methods currently employed do not give any information. For this reason, there is a growing interest towards the development of liquid chromatographic methods for the enantioseparation of cannabinoids. Much work is needed in this field where one of the major limitations is the lack of optically pure standards. This manuscript reports about the chromatographic behavior of five popular cannabinoids (including the cannabichromene racemate, CBC) on nine immobilised polysaccharide-based chiral stationary phases (CSPs) differently substituted, under reversed phase conditions. Results showed that chemo-selectivity of CSPs is not affected by changes in mobile phase composition, in the range of mobile phase investigated. In addition, the presence of electron withdrawing groups on the CSPs systematically leads to shorter retention times compared to when electron donating groups are present. An application of separation of cannabinoids from a real hemp extract on two of the chiral columns employed in this work revealed the presence of both CBC enantiomers in the sample.