Liquid Chromatographic Separation Research Articles

Investigation of the phytochemical profiling and antioxidant, anti-diabetic, anti-inflammatory, and MDA-MB-231 cell line antiproliferative potentials of extracts from Ephedra alata Decne

Ephedra is one of the many medicinal herbs that have been used as folk/traditional medicine in Jordan and other countries to cure various illnesses. Plants of this genus are well known for their antioxidant and antibacterial properties. In this study, three different solvents were used to obtain Ephedra extracts. When evaluated, the Ephedra alata Decne ethanolic extract reportedly had the greatest levels of total phenolic compounds (TPC) and total flavonoid compounds (TFC). The aqueous extracts displayed the highest antioxidant activity in the DPPH and ABTS assays, demonstrating their considerable capacity to neutralize free radicals. However, when evaluated using the FRAP method, the acetone extracts showed the strongest antioxidant activity, indicating their high reducing power. LC–MS/MS, a potent method of analysis that combines the liquid chromatographic separation properties with mass spectrometry detection and identification capabilities, was used in this study to detect and measure phytochemical content of a total of 24 phenolic compounds and 16 terpene compounds present in the extracts of Ephedra alata Decne. Various concentrations of these chemicals were found in these extracts. The extracts’ inhibitory effects on albumin denaturation and alpha-amylase activity were also assessed; the findings demonstrated the potentials of these extracts as anti-inflammatory and anti-diabetic medicines, with the acetone extract having the lowest IC50 values in the concomitant tests (306.45 µg/ml and 851.23 µg/ml, respectively). Furthermore, the lowest IC50 value (of 364.59 ± 0.45 µg/ml) for the 80% ethanol extract demonstrated that it has the strongest antiproliferative impact regarding the MDA-MB-231 breast cancer cell line. This finding indicates that this particular extract can be potentially used to treat cancer.

Functional Insights into the Sphingolipids C1P, S1P, and SPC in Human Fibroblast-like Synoviocytes by Proteomic Analysis

The (patho)physiological function of the sphingolipids ceramide-1-phosphate (C1P), sphingosine-1-phosphate (S1P), and sphingosylphosphorylcholine (SPC) in articular joints during osteoarthritis (OA) is largely unknown. Therefore, we investigated the influence of these lipids on protein expression by fibroblast-like synoviocytes (FLSs) from OA knees. Cultured human FLSs (n = 7) were treated with 1 of 3 lipid species—C1P, S1P, or SPC—IL-1β, or with vehicle. The expression of individual proteins was determined by tandem mass tag peptide labeling followed by high-resolution electrospray ionization (ESI) mass spectrometry after liquid chromatographic separation (LC-MS/MS/MS). The mRNA levels of selected proteins were analyzed using RT-PCR. The 3sphingolipids were quantified in the SF of 18 OA patients using LC-MS/MS. A total of 4930 proteins were determined using multiplex MS, of which 136, 9, 1, and 0 were regulated both reproducibly and significantly by IL-1β, C1P, S1P, and SPC, respectively. In the presence of IL-1ß, all 3 sphingolipids exerted ancillary effects. Only low SF levels of C1P and SPC were found. In conclusion, the 3 lipid species regulated proteins that have not been described in OA. Our results indicate that charged multivesicular body protein 1b, metal cation symporter ZIP14, glutamine-fructose-6-P transaminase, metallothionein-1F and -2A, ferritin, and prosaposin are particularly interesting proteins due to their potential to affect inflammatory, anabolic, catabolic, and apoptotic mechanisms.

Performance of global retention models in the optimisation of the liquid chromatographic separation (II): Complex multi-analyte samples

BackgroundEnhancing the quality control of medicinal plants is a complex challenge due to their rich variety of chemical compounds present at varying and extreme concentrations. Chromatographic fingerprints, which have become essential for characterising these complex natural materials, require achieving optimal separation conditions to effectively maximise the number of detected peaks. The challenges in optimising fingerprints and other complex multi-analyte samples include the unavailability of standards, the presence of unknown constituents and the substantial workload that would require conventional optimisation methods based on models. ResultsThis work introduces an interpretive optimisation approach which operates on the premise of predicting chromatograms using global models. Initially, a multi-linear gradient experimental design is sequentially executed to accommodate all peaks in the chromatogram in an adequate time window. Following this, a small set of sample peaks (reference peaks) is selected based on their consistent traceability across all chromatograms in the design. Using this reference dataset, a global model is constructed, initially focused solely on the reference peaks and later extended to encompass all detected peaks in the sample. The aim is to find gradients that maximise resolution while minimising analysis time. These optimised gradients are applied successfully to enhance the separation of medicinal plant extracts, with particular emphasis on peppermint and pennyroyal extracts. SignificanceThe proposed optimisation relying on global models can be applied to highly complex samples even in the absence of standards, or in cases where standards are available but their use is impractical due to workload constraints. Moreover, in discerning the most promising gradients for highly complex samples, peak purity has demonstrated superior reliability and competitiveness compared to peak capacity as chromatographic objective function.

PeakQC: A Software Tool for Omics-Agnostic Automated Quality Control of Mass Spectrometry Data.

Mass spectrometry is broadly employed to study complex molecular mechanisms in various biological and environmental fields, enabling 'omics' research such as proteomics, metabolomics, and lipidomics. As study cohorts grow larger and more complex with dozens to hundreds of samples, the need for robust quality control (QC) measures through automated software tools becomes paramount to ensure the integrity, high quality, and validity of scientific conclusions from downstream analyses and minimize the waste of resources. Since existing QC tools are mostly dedicated to proteomics, automated solutions supporting metabolomics are needed. To address this need, we developed the software PeakQC, a tool for automated QC of MS data that is independent of omics molecular types (i.e., omics-agnostic). It allows automated extraction and inspection of peak metrics of precursor ions (e.g., errors in mass, retention time, arrival time) and supports various instrumentations and acquisition types, from infusion experiments or using liquid chromatography and/or ion mobility spectrometry front-end separations and with/without fragmentation spectra from data-dependent or independent acquisition analyses. Diagnostic plots for fragmentation spectra are also generated. Here, we describe and illustrate PeakQC's functionalities using different representative data sets, demonstrating its utility as a valuable tool for enhancing the quality and reliability of omics mass spectrometry analyses.

Exploratory Mass Spectrometry of Cerebrospinal Fluid from Persons with Autopsy-Confirmed LATE-NC.

Common neuropathologies associated with dementia include Alzheimer's disease neuropathologic change (ADNC) and limbic-predominant age-related TDP-43 encephalopathy neuropathologic change (LATE-NC). Biofluid proteomics provides a window into the pathobiology of dementia and the information from biofluid tests may help guide clinical management. Participants (n = 29) had been autopsied and had antemortem CSF draws in a longitudinal cohort of older adults at the University of Kentucky AD Research Center. Cases were designated as LATE-NC + if they had LATE-NC stage > 1 (n = 9); the remaining 20 cases were designated LATE-NC-. This convenience sample of CSF specimens was analyzed in two separate processes: From one group, aliquots were depleted of highly abundant proteins using affinity spin columns. Tryptic digests of sample proteins were subjected to liquid chromatographic separation and mass spectrometry. Relative quantification was performed using Sciex software. Peptides referent to a total of 949 proteins were identified in the samples depleted of abundant proteins, and 820 different proteins were identified in the non-depleted samples. When the Bonferroni/false-discovery statistical correction was applied to account for having made multiple comparison tests, only 4 proteins showed differential expression (LATE-NC + vs LATE-NC-) in the non-depleted samples (RBP4, MIF, IGHG3, and ITM2B). Post hoc western blots confirmed that RBP4 expression was higher in the LATE-NC + cases at the group level. In summary, an exploratory assessment of proteomes of autopsy-confirmed LATE-NC and non-LATE-NC CSF did not demonstrate a clear-cut proteomic fingerprint that distinguished the two groups. There was, however, an increase in RBP4 protein levels in CSF from LATE-NC cases.

Exploratory Mass Spectrometry of Cerebrospinal Fluid from Persons with Autopsy-Confirmed LATE-NC.

Common neuropathologies associated with dementia include Alzheimer's disease neuropathologic change (ADNC) and limbic-predominant age-related TDP-43 encephalopathy neuropathologic change (LATE-NC). Biofluid proteomics provides a window into the pathobiology of dementia and the information from biofluid tests may help guide clinical management. Participants (n = 29) had been autopsied and had antemortem CSF draws in a longitudinal cohort of older adults at the University of Kentucky AD Research Center. Cases were designated as LATE-NC + if they had LATE-NC stage > 1 (n = 9); the remaining 20 cases were designated LATE-NC-. This convenience sample of CSF specimens was analyzed in two separate processes: From one group, aliquots were depleted of highly abundant proteins using affinity spin columns. Tryptic digests of sample proteins were subjected to liquid chromatographic separation and mass spectrometry. Relative quantification was performed using Sciex software. Peptides referent to a total of 949 proteins were identified in the samples depleted of abundant proteins, and 820 different proteins were identified in the non-depleted samples. When the Bonferroni/false-discovery statistical correction was applied to account for having made multiple comparison tests, only 4 proteins showed differential expression (LATE-NC + vs LATE-NC-) in the non-depleted samples (RBP4, MIF, IGHG3, and ITM2B). Post hoc western blots confirmed that RBP4 expression was higher in the LATE-NC + cases at the group level. In summary, an exploratory assessment of proteomes of autopsy-confirmed LATE-NC and non-LATE-NC CSF did not demonstrate a clear-cut proteomic fingerprint that distinguished the two groups. There was, however, an increase in RBP4 protein levels in CSF from LATE-NC cases.

The optimised method of HPLC analysis of glutathione allows to determine the degree of oxidative stress in plant cell culture

Redox regulations and antioxidant defence play a central role in the acclimation of plants to their environment. Glutathione represents an essential component of the cellular antioxidant defence system, which keeps levels of reactive oxygen species (ROS) under control. High-performance liquid chromatography (HPLC) separation with fluorescence detection is a sensitive method that enables analysis of reduced and oxidised glutathione levels in small samples of plant tissues or plant cell culture. We aimed to optimise the method to obtain more accurate information about the total level of glutathione and the proportion of the reduced form (GSH) by choosing the most suitable reduction reagent and the conditions under which the reduction occurs. The applicability of the developed method was verified by analysing tobacco cells treated with hydrogen peroxide, which caused a decrease in the GSH/total glutathione ratio. Significant changes in the level of glutathione as well as in the GSH/total glutathione ratio were also observed during tobacco cell culture development.

Recent progress for chiral stationary phases based on chiral porous materials in high-performance liquid chromatography and gas chromatography separation.

Chirality is a fundamental property of nature. Separation and analysis of racemates are of great importance in the fields of medicine and the production of chiral biopharmaceutical intermediates. Chiral chromatography has the characteristics of a wide separation range, fast separation speed, and high efficiency. The development and preparation of novel chiral stationary phases with good chiral recognition and separation capacity is the core and key of chiral chromatographic separation and analysis. In this work, the representative research progress of novel chiral porous crystal materials including chiral covalent organic frameworks, chiral porous organic cages, chiral metal-organic frameworks, and chiral metal-organic cages used as chiral stationary phases of capillary gas chromatography and high-performance liquid chromatography over the last 4 years is reviewed in detail. The chiral recognition and separation properties of the representative studies in this review are also introduced and discussed.

Skeletal Muscle Proteome Modifications following Antibiotic-Induced Microbial Disturbances in Cancer Cachexia.

Cancer cachexia is an involuntary loss of body weight, mostly of skeletal muscle. Previous research favors the existence of a microbiota-muscle crosstalk, so the aim of the study was to evaluate the impact of microbiota alterations induced by antibiotics on skeletal muscle proteins expression. Skeletal muscle proteome changes were investigated in control (CT) or C26 cachectic mice (C26) with or without antibiotic treatment (CT-ATB or C26-ATB, n = 8 per group). Muscle protein extracts were divided into a sarcoplasmic and myofibrillar fraction and then underwent label-free liquid chromatography separation, mass spectrometry analysis, Mascot protein identification, and METASCAPE platform data analysis. In C26 mice, the atrogen mafbx expression was 353% higher than CT mice and 42.3% higher than C26-ATB mice. No effect on the muscle protein synthesis was observed. Proteomic analyses revealed a strong effect of antibiotics on skeletal muscle proteome outside of cachexia, with adaptative processes involved in protein folding, growth, energy metabolism, and muscle contraction. In C26-ATB mice, proteome adaptations observed in CT-ATB mice were blunted. Differentially expressed proteins were involved in other processes like glucose metabolism, oxidative stress response, and proteolysis. This study confirms the existence of a microbiota-muscle axis, with a muscle response after antibiotics that varies depending on whether cachexia is present.

HPLC-DAD method for concurrent estimation of netarsudil, latanoprost and benzalkonium chloride: Stability indicating study, impurity separation, assay of ophthalmic preparations and greenness/whiteness valuation

A validated, stability indicating, multiuse and sustainable HPLC-DAD method was developed for simultaneous determination and separation of the ophthalmic ternary mixture of netarsudil (NTS), latanoprost (LTP) and benzalkonium chloride (BZC) in presence of four related compounds and suspected impurities. Effectual liquid chromatographic separation was accomplished by using a Zorbax-SB Phenyl column with gradient elution of a mobile phase consisting of buffer mixture solution of phosphate and sodium pentane sulfonate (pH 3.0) and acetonitrile at flow rate 1.5 mL/min. DAD was tuned at 254 nm for measuring peak area of NTS and 210 nm for LTP and BZC. The proposed procedure showed to be effective in the stability investigation of the cited drugs. The suggested HPLC procedure was validated based on the International Council for Harmonization (ICH) guiding principles. The three analytes were determined within linearity ranges of 1–100 µg/mL, 5–200 µg/mL and 10–200 µg/mL for NTS, LTP and BZC respectively with excellent analytical performance. Assay of several ophthalmic preparations was successfully carried out using the validated method, which was further appraised together with some relevant reported methods in regard to green and white analytical chemistry aspects using AGREE and RGB-12 tools. Very few published analytical records were found for NTS/LTP mixture. After surveying the scientific literature, no documentation was retrieved for a similar inclusive stability-indicating impurity-profiling separative method for the synchronized quantitation of NTS, LTP together with co-formulated preservative BZC.

Establishment of an at-line nanofractionation-based screening platform by coupling HPLC-MS/MS with high-throughput fluorescence polarization bioassay for natural SARS-CoV-2 fusion inhibitors

In this study, a novel at-line nanofractionation platform was established for screening SARS-CoV-2 fusion inhibitors from natural products for the first time by combining HPLC-MS/MS with high-throughput fluorescence polarization (FP) bioassay. A time-course FP bioassay in 384 well-plates was conducted in parallel with MS/MS to simultaneously obtain chemical and biological information of potential fusion inhibitors in Lonicerae Japonicae Flos (LJF) and Lianhua Qingwen capsules (LHQW). Semi-preparative liquid chromatography and orthogonal HPLC separation were employed to enrich and better identify the co-eluted components. After comprehensive evaluation and validation, 28 potential SARS-CoV-2 fusion inhibitors were screened out and identified. Several compounds at low micromolar activity were validated by in vitro inhibitory assay, molecular docking, cytotoxicity test, and pseudovirus assay. Moreover, four potential dual-target inhibitors against influenza and COVID-19 were discovered from LJF using this method, offering novel insights for the development of future pharmaceuticals targeting epidemic respiratory diseases.

Two-Dimensional Thin-Layer Chromatography as an Accessible, Low-Cost Tool for Lipid-Class Profile Screening

The interest in lipid composition profiling is significantly increasing as research reveals the immense importance of lipids in medicine, plant science, food and agriculture. However, lipidomic analysis requires high-end specialty equipment. We used two-dimensional thin-layer chromatography (2D-TLC) as a readily available, low-cost tool for basic lipidomic profiling of lipid classes in algal samples in the models Chlamydomonas reinhardtii, Auxenochlorella protothecoides, and Euglena gracilis, validating lipid class identification using an LC-MS/MS analysis. Algal lipid extracts were separated on a 2D-TLC plate, and TLC analysis was followed by scraping individual TLC spots off the plate, and a subsequent liquid chromatography separation and tandem mass spectrometry (LC-MS/MS) analysis. For comparison, crude lipid extracts were also injected directly to the LC-MS/MS system. Lipid class annotation was achieved by a combination of accurate mass, retention time information, neutral loss and fragment ion analysis by MS2Analyzer, and by matching spectra to LipidBlast MS/MS library. Overall, we were able to identify 15 lipid classes, and to adequately profile the lipid classes in all three organisms. This TLC method is thus suggested as an accessible tool for lipid class profiling of algal, plant, and food lipids, alike, when a rapid and simple analysis is required, e.g., for screening purposes.

Research Progress in the Separation of Chemical Components from Essential Oils by High-Speed Countercurrent Chromatography

Essential oils (EOs) are vital secondary metabolites in plants. They have garnered substantial attention owing to their distinct flavors and desirable attributes, including potent antioxidant, antibacterial, and antitumor properties. Nevertheless, the active constituents of EOs exhibit intricate chemical structures, and conventional separation techniques are inadequate for purifying the individual chemical components from EOs. High-speed countercurrent chromatography, based on the principles of a hydrodynamic equilibrium system, has emerged as a liquid–liquid chromatographic separation method renowned for its ability to handle substantial single injection volumes and the absence of irreversible adsorption. Consequently, in recent years, this technique has been widely employed in the isolation and refinement of natural products. In this review, a comprehensive analysis is conducted, contrasting the merits and demerits of high-speed countercurrent chromatography with conventional separation methods. The solvent systems, elution modes, commonly employed detectors, and practical applications are reviewed in the context of high-speed countercurrent chromatography for essential oil separation and purification. Furthermore, this review offers a glimpse into the potential prospects of applying this technique, with the intention of serving as a valuable reference for the use of high-speed countercurrent chromatography in the purification of EOs.

Choline chloride-L-lactic acid mixtures as solvents for extraction of Coumarin from cinnamon-containing foods

The determination of coumarin in generally available cinnamon-containing foods such as biscuits, cereals is important to ensure that the populations consuming these foods more frequently are not exposed to higher levels and are not at risk. This study describes the development of an analytical method for determination of coumarin in foods containing cinnamon. Solid-liquid phase extraction with conventional stirring-assisted and ultrasound-assisted extraction (UAE) procedures were used to prepare sample extracts for liquid chromatography analysis. The green solvent, a mixture of choline chloride:L-lactic acid (1:4, mol/mol)/water (50:50 v/v), as a deep eutectic solvent-like mixture, was selected as an extraction solvent. The extraction conditions were optimized by using the design of the experiment strategy obtaining parameters, temperature 47 °C, time 36 min, solvent volume 4 mL, sample amount 0.8 g, stirring speed 20 rpm for rotary stirring-assisted extraction procedure, and temperature 30 °C, time 5 min, solvent volume 3.5 mL, sample amount 0.8 g for UAE. High-performance liquid chromatography separation with a total analysis time of 12 min was performed with a stationary phase of octadecyl type and a gradient of mobile phase of methanol-1 % acetic acid. The methods were successfully validated to show the detection limit of 0.13 μg.mL−1, the linearity range of 0.4–50 µg.mL−1 (R2 = 0.999), recovery values more than 80 %, and the RSDs of less than 7 %. Rotary stirring-assisted extraction showed moderately higher recovery values than UAE, while lower RSD % was observed for UAE.

Synergistic expression of ZIF-67 and amphiphilic polymer hydrogel in multi-mode liquid chromatographic separation

In this work, zeolitic imidazolate framework-67 (ZIF-67) with excellent stability synthesized at room temperature was used together with amphiphilic polymer hydrogel to synthesize a captivating mixed-mode silica stationary phase for the first time. The amphiphilic polymer hydrogel prepared by weakly basic cationic and acidic anionic monomers that was modified on the surface of ZIF-67@SiO2 by a simple physical coating method not only improved the separation performance, but also enhanced the applicability of ZIF-67@SiO2 in chromatographic analysis. Meanwhile, the porous ZIF-67 effectively alleviated the problem that polymer hydrogel was easy to swell. To sum up, the amphiphilic polymer hydrogel and ZIF-67 showed excellent synergistic effect in improving the separation ability and the applicability of stationary phase. The experimental results demonstrated that the ZIF-67/hydrogel@SiO2 stationary phase could be applied in various chromatographic separation modes, demonstrating superior resolution and separation selectivity for polycyclic aromatic hydrocarbons, steroid hormones, nucleosides, B-vitamins, sulfonamides, benzoic acids and dinitrobenzene isomers. In this study, the synergistic expression of ZIF-67 and polymer hydrogel provided a new idea for the development of silica stationary phase co-modified by metal-organic framework and hydrogel material. Concurrently, the successful preparation of ZIF-67/hydrogel@SiO2 composite revealed a new possibility for utilizing ZIF-67 as the functional material of stationary phase, expanding the application potential of ZIF-67 in the field of chromatography.

Occurrence, exposure estimation, and dietary risk assessment of biogenic amines in tea samples marketed in Malaysia

Biogenic amines (BAs) are one of the most studied food contaminants due to their widespread distribution in food matrices. However, the occurrence and dietary risks of BAs in tea, particularly in Malaysia, have not been comprehensively investigated. This study evaluates the occurrence of BAs in selected tea samples from Malaysia's market, and the estimation of the dietary exposure to assess the risk of their dietary intake. The targeted BAs (tyramine, histamine, tryptamine, spermine, spermidine, putrescine, and cadaverine) were extracted with 0.1 M HCl using ultrasound, followed by derivatization using dansyl chloride prior to high performance liquid chromatographic separation. The validated method was applied to analyzing BAs in various tea samples of different origins. Results showed that the occurrence of histamine (14.3–69.5 μg g−1) in fully fermented tea, semi-fermented tea, non-fermented tea, post-fermented tea, and tisanes was 8, 0, 63, 17, and 0%, respectively. Spermidine was found in all samples (except 2 tisanes) with concentrations ranging from 17.6 to 105.4 μg g−1. Tyramine was identified as the most prevalent BA (7.2–1271.7 μg g−1), with a detection frequency of 90.2%. Overall, the average total BAs content in fully fermented tea (284.2 μg g−1), semi-fermented tea (677.0 μg g−1), non-fermented tea (783.8 μg g−1), post-fermented tea (204.6 μg g−1), and tisanes (134.2 μg g−1) were below the recommended level of 1000 μg g−1. Dietary exposure was determined by estimated daily intake, whereas risk assessment was evaluated using the hazard quotient (HQ) and hazard index (HI) approaches. Based on the obtained HQ (≤0.017322) and HI (≤0.033437), the dietary exposure to BAs through the consumption of tea in Malaysia does not pose potential health risks to consumers.

A new green approach to synthesizing MIP-202@porous silica microspheres for positional isomer/enantiomer/hydrophilic separation

Metal-organic frameworks (MOFs) with superior physicochemical properties have great potential for applications in chromatographic separation. However, currently popular methods for the synthesis of MOF-based silica composite materials usually require the use of harmful organic solvents and long-term high-temperature sealing reactions. In order to respond to the needs of green chromatography, it is urgent to develop a new green organic-solvent-free strategy for the synthesis of MOF@SiO2 composites. MIP-202 is a zirconium-MOF constructed from zirconium ion and L-aspartic acid, which features green synthesis as well as good hydrolytic stability and chemical stability. In this paper, SiO2-NH2 was first prepared in a hydrophilic deep eutectic solvent, and then an amino acid-based MOF material (MIP-202) was modified on the surface of the SiO2-NH2 in an aqueous solution to obtain a MIP-202@SiO2 composite material. The multi-mode separation performance of MIP-202@SiO2 as a promising liquid chromatographic stationary phase was particularly evaluated and the separation mechanisms were discussed. The MIP-202@SiO2 column exhibited excellent separation ability for aromatic positional isomers. In addition, chiral enantiomers and hydrophilic analytes were also satisfactorily detected and separated. This work provides a new approach for the facile synthesis of MOF-based liquid chromatographic separation material by using green deep eutectic solvent and water as the reaction media.

The identification of psychedelic compounds within Australian Acacia species

ABSTRACT N,N-dimethyltryptamine (DMT) is a psychedelic substance produced within various Acacia species; however, the range of species that contain it and the quantity of the compound found within each species is relatively unknown. This work focused on the identification of DMT in 104 plant samples, representing 79 Acacia species, utilizing a reverse phase liquid chromatographic separation prior to mass spectral characterization with a robust Q-TOF-MS method. The findings of this work determined 51 DMT-containing species, 41 of which were previously unknown to contain the psychedelic.

The Impact of Different Drying Methods on the Metabolomic and Lipidomic Profiles of Arthrospira platensis.

Drying is an inseparable part of industrial microalgae production. In this work, the impacts of eight different drying methods on the metabolome and lipidome of Arthrospira platensis were investigated. The studied drying methods were freeze drying (FD), sun drying (SD), air drying at 40 and 75 °C (AD' and AD″), infrared drying at 40 and 75 °C (IRD' and IRD″), and vacuum drying at 40 and 75 °C (VD' and VD″). Results gathered by reversed-phase liquid chromatography separation coupled with high-resolution tandem mass spectrometry with electrospray ionization (RP-LC-ESI-Orbitrap HRMS/MS) analysis allowed researchers to identify a total of 316 metabolites (including lipids) in aqueous and ethanolic extracts. The compounds identified in ethanolic extracts were mainly lipids, such as neutral and polar lipids, chlorophylls and carotenoids, while the compounds identified in the aqueous extracts were mainly amino acids and dipeptides. Among the identified compounds, products of enzymatic and chemical degradation, such as pyropheophytins, monoacylglycerols and lysophosphatidylcholines were also identified and their amounts depended on the drying method. The results showed that except for FD method, recognized as a control, the most protective method was AD'. Contrary to this, VD' and VD″, under the conditions used, promoted the most intense degradation of valuable metabolites.

A Study of Metabolites from Basidiomycota and Their Activities against Pseudomonas aeruginosa.

The World Health Organization (WHO) promotes research aimed at developing new drugs from natural compounds. Fungi are important producers of bioactive molecules, and they are often effective against other fungi and/or bacteria and are thus a potential source of new antibiotics. Basidiomycota crude extracts, which have previously been proven to be active against Pseudomonas aeruginosa ATCC27853, were subjected to liquid chromatographic separation by RP-18, leading to six macro-fractions for each fungal extract. The various fractions were tested for their bioactivities against P. aeruginosa ATCC27853, and ten of them were characterized by HPLC-HRMS and NMR. Further chromatographic separations were performed for a few selected macro-fractions, yielding seven pure compounds. Bioactivity was mainly found in the lipophilic fractions containing fatty acids and their derivatives, such as hydroxy or keto C-18 unsaturated acids, and in various complex lipids, such as glycolipids and related compounds. More hydrophilic molecules, such as GABA, phenethylamine, two chromogenic anthraquinoids and pistillarin, were also isolated, and their antibacterial activities were recorded. The novelties of this research are as follows: (i) the genera Cortinarius and Mycena have never been investigated before for the synthesis of antibiotic compounds; (ii) the molecules produced by these genera are known, but their production has never been reported in the investigated fungi; (iii) the determination of bacterial siderophore synthesis inhibition by certain compounds from Cortinarius and Mycena.