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.

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.

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.

Composition and nutritional values of fatty acids in marine organisms by one-step microwave-assisted extraction/derivatization and comprehensive two-dimensional gas chromatography -flame ionization detector.

This work reports the characterization of the lipidic fraction of seven species of marine organisms gathered along the shoreline of the Po Delta Park of Emilia-Romagna Region (Italy) and of the north Adriatic Sea. Two species of oysters (Crassostrea gigas and Ostrea edulis), two species of clams (Chamelea gallina and Ruditapes philippinarum), one species of mussel (Mytilus galloprovincialis), one species of macroalgae (Ulva rigida), and one species of spiny dogfish (Squalus acanthias) were analyzed to characterize their fatty acids profile and related nutritional value. The lipid fraction was simultaneously extracted and transesterified into fatty acid methyl esters (FAMEs) by using a recently developed one-step microwave-assisted extraction/derivatization (MAED) method. The obtained FAMEs extract was analyzed by a rapid comprehensive multidimensional gas chromatography (GC × GC) method (30 min). The system was equipped with a reverse set of columns (polar × non-polar) connected through a reversed fill/flush flow modulator. The GC × GC system was coupled with a flame-ionization detector (FID) for both qualitative and quantitative purposes. The MAED- GC × GC-FID methodology was suitable in the context of samples containing high percentages of omega-3 PUFA. A total of 82 FAMEs were tentatively identified using standards, literature data, and the two-dimensional plot location. FAME profiles obtained with the proposed approach were comparable with reference methods (AOCS Ce 2b-11), showing no significant differences. Moreover, to determine the food nutritional value of the samples investigated, the most common nutritional indices (index of atherogenicity, index thrombogenicity, hypocholesterolemic/hypercholesterolemic ratio, health-promoting index, unsaturation index, and the fish lipid quality index) were calculated from FAME profiles. Among the samples investigated, Squalus acanthias presented the best nutritional score, while Ruditapes philippinarum had the worst score in 3 out of 6 indices.

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.

miRNAs as Biomolecular Markers for Food Safety, Quality, and Traceability in Poultry Meat-A Preliminary Study.

In this study, the expression and abundance of two candidate chicken (Gallus gallus; gga) microRNAs (miRNAs, miR), gga-miR-21-5p (miR-21) and gga-miR-126-5p (miR-126), have been analyzed in order to identify biomarkers for the traceability and quality of poultry meat. Two breeds of broiler chickens were tested: the most common Ross308 (fast-growing) and the high-quality Ranger Gold (slow-growing). A preliminary analysis of the two miRNAs expressions was conducted across various tissues (liver, lung, spleen, skeletal muscle, and kidney), and the three tissues (lung, spleen, and muscle) with a higher expression were chosen for further analysis. Using quantitative reverse transcription polymerase chain reaction (RT-qPCR), the expression of miRNAs in the three tissues of a total of thirteen animals was determined. The results indicate that miR-126 could be a promising biomarker for the lung tissue in the Ranger Gold (RG) breed (p < 0.01), thus suggesting a potential applicability for tracing hybrids. RG exhibits a significantly higher miR-126 expression in the lung tissue compared to the Ross308 broilers (R308), an indication of greater respiratory capacity and, consequently, a higher oxidative metabolism of the fast-growing hybrid. During sampling, two R308 broilers presented some anomalies, including airsacculitis, hepatic steatosis, and enlarged spleen. The expression of miR-126 and miR-21 was compared in healthy animals and in those presenting anomalies. Chickens with airsacculitis and hepatic steatosis showed an up-regulation of miR-21 and miR-126 in the most commercially valuable tissue, the skeletal muscle or breast (p < 0.05).

One-phase extraction coupled with photochemical reaction allows the in-depth lipid characterization of hempseeds by untargeted lipidomics.

Due to their valuable nutritional content, several hemp-derived products from hempseeds have recently been placed in the market as food and food ingredients. In particular, the lipid composition of hempseeds has raised interest for their rich content in biologically active polyunsaturated fatty acids with an optimum ratio of omega-3 and omega-6 compounds. At present, however, the overall polar lipidome composition of hempseeds remains largely unknown. In the present work, an analytical platform was developed for the extraction, untargeted HRMS-based analysis, and detailed annotation of the lipid species. First, five one- and two-phase solid-liquid extraction protocols were tested and compared on a hempseed pool sample to select the method that allowed the overall highest efficiency as well as easy coupling with lipid derivatization by photochemical [2 + 2] cycloaddition with 6-azauracil. Underivatized lipids were annotated employing a data processing workflow on Compound Discoverer software that was specifically designed for polar lipidomics, whereas inspection of the MS/MS spectra of the derivatized lipids following the aza-Paternò-Büchi reaction allowed pinpointing the regiochemistry of carbon-carbon double bonds. A total of 184 lipids were annotated, i.e., 26 fatty acids and 158 phospholipids, including minor subclasses such as N-acylphosphatidylethanolamines. Once the platform was set up, the lipid extracts from nine hempseed samples from different hemp strains were characterized, with information on the regiochemistry of free and conjugated fatty acids. The overall analytical approach helped to fill a gap in the knowledge of the nutritional composition of hempseeds.

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.

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.

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.

Enantioseparation of chiral phytocannabinoids in medicinal cannabis.

The evaluation of the chiral composition of phytocannabinoids in the cannabis plant is particularly important as the pharmacological effects of the (+) and (-) enantiomers of these compounds are completely different. Chromatographic attempts to assess the presence of the minor (+) enantiomers of the main phytocannabinoids, cannabidiolic acid (CBDA) and trans-Δ9-tetrahydrocannabinolic acid (trans-Δ9-THCA), were carried out on heated plant extracts for the determination of the corresponding decarboxylated species, cannabidiol (CBD) and trans-Δ9-tetrahydrocannabinol (trans-Δ9-THC), respectively. This process produces an altered phytocannabinoid composition with several new and unknown decomposition products. The present work reports for the first time the stereoselective synthesis of the pure (+) enantiomers of the main phytocannabinoids, trans-CBDA, trans-Δ9-THCA, trans-CBD and trans-Δ9-THC, and the development and optimization of an achiral-chiral liquid chromatography method coupled to UV and high-resolution mass spectrometry detection in reversed phase conditions (RP-HPLC-UV-HRMS) for the isolation of the single compounds and evaluation of their actual enantiomeric composition in plant. The isolation of the peaks with the achiral stationary phase ensured the absence of interferences that could potentially co-elute with the analytes of interest in the chiral analysis. The method applied to the Italian medicinal cannabis variety FM2 revealed no trace of the (+) enantiomers for all phytocannabinoids under investigation before and after decarboxylation, thus suggesting that the extraction procedure does not lead to an inversion of configuration.

Current Developments of Analytical Methodologies for Aflatoxins' Determination in Food during the Last Decade (2013-2022), with a Particular Focus on Nuts and Nut Products.

This review aims to provide a clear overview of the most important analytical development in aflatoxins analysis during the last decade (2013-2022) with a particular focus on nuts and nuts-related products. Aflatoxins (AFs), a group of mycotoxins produced mainly by certain strains of the genus Aspergillus fungi, are known to impose a serious threat to human health. Indeed, AFs are considered carcinogenic to humans, group 1, by the International Agency for Research on Cancer (IARC). Since these toxins can be found in different food commodities, food control organizations worldwide impose maximum levels of AFs for commodities affected by this threat. Thus, they represent a cumbersome issue in terms of quality control, analytical result reliability, and economical losses. It is, therefore, mandatory for food industries to perform analysis on potentially contaminated commodities before the trade. A full perspective of the whole analytical workflow, considering each crucial step during AFs investigation, namely sampling, sample preparation, separation, and detection, will be presented to the reader, focusing on the main challenges related to the topic. A discussion will be primarily held regarding sample preparation methodologies such as partitioning, solid phase extraction (SPE), and immunoaffinity (IA) related methods. This will be followed by an overview of the leading analytical techniques for the detection of aflatoxins, in particular liquid chromatography (LC) coupled to a fluorescence detector (FLD) and/or mass spectrometry (MS). Moreover, the focus on the analytical procedure will not be specific only to traditional methodologies, such as LC, but also to new direct approaches based on imaging and the ability to detect AFs, reducing the need for sample preparation and separative techniques.

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.

The contribution of high-resolution GC separations in plastic recycling research.

One convenient strategy to reduce environmental impact and pollution involves the reuse and revalorization of waste produced by modern society. Nowadays, global plastic production has reached 367 million tons per year and because of their durable nature, their recycling is fundamental for the achievement of the circular economy objective. In closing the loop of plastics, advanced recycling, i.e., the breakdown of plastics into their building blocks and their transformation into valuable secondary raw materials, is a promising management option for post-consumer plastic waste. The most valuable product from advanced recycling is a fluid hydrocarbon stream (or pyrolysis oil) which represents the feedstock for further refinement and processing into new plastics. In this context, gas chromatography is currently playing an important role since it is being used to study the pyrolysis oils, as well as any organic contaminants, and it can be considered a high-resolution separation technique, able to provide the molecular composition of such complex samples. This information significantly helps to tailor the pyrolysis process to produce high-quality feedstocks. In addition, the detection of contaminants (i.e., heteroatom-containing compounds) is crucial to avoid catalytic deterioration and to implement and design further purification processes. The current review highlights the importance of molecular characterization of waste stream products, and particularly the pyrolysis oils obtained from waste plastics. An overview of relevant applications published recently will be provided, and the potential of comprehensive two-dimensional gas chromatography, which represents the natural evolution of gas chromatography into a higher-resolution technique, will be underlined.

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.