Electrospray Research Articles

Infrared Photoactivation Enables nano-DESI MS of Protein Complexes in Tissue on a Linear Ion Trap Mass Spectrometer.

Native mass spectrometry analysis of proteins directly from tissues can be performed by using nanospray-desorption electrospray ionization (nano-DESI). Typically, supplementary collisional activation is essential to decluster protein complex ions from solvent, salt, detergent, and lipid clusters that comprise the ion beam. As an alternative, we have implemented declustering by infrared (IR) photoactivation on a linear ion trap mass spectrometer equipped with a CO2 laser (λ = 10.6 μm). The prototype system demonstrates declustering of intact protein complex ions up to approximately 50 kDa in molecular weight that were sampled directly from brain and eye lens tissues by nano-DESI. For example, signals for different metal binding states of hSOD1G93A homodimers (approximately 32 kDa) separated by only approximately 6 Th (10+ ions) were resolved with IR declustering, but not with collisional activation. We found IR declustering to outperform collisional activation in its ability to reduce chemical background attributable to nonspecific clusters in the nano-DESI ion beam. The prototype system also demonstrates in situ native MS on a low-cost mass spectrometer and the potential of linear ion trap mass spectrometers for this type of analysis.

Intermolecular Interactions between Aromatic Amino Acids Investigated by Ultraviolet Photodissociation Spectroscopy of Hydrogen-Bonded Clusters of Histidine and Tryptophan Enantiomers.

Intermolecular interactions between aromatic amino acids were investigated by ultraviolet photodissociation spectroscopy of hydrogen-bonded protonated clusters of histidine (His) and tryptophan (Trp) enantiomers in the gas phase. Product ion spectra and photodissociation spectra in the wavelength range of the S1-S0 transition of Trp at several temperatures (8-100 K) were obtained using a tandem mass spectrometer equipped with an electrospray ionization source and a cold ion trap. l-Trp detachment forming protonated His was the main pathway. Two bands observed at 288 and 285 nm in the photodissociation spectra of heterochiral H+(d-His)(l-Trp) indicated the coexistence of two types of conformers. The bands at 288 and 285 nm were attributed to the conformers formed from stronger and weaker intermolecular interactions, respectively. In the spectra of homochiral H+(l-His)(l-Trp), only the band due to the stronger interactions was observed at 288 nm. The intermolecular interactions of l-His with l-Trp were stronger than those of d-His with l-Trp.

Chemical modification of selenium-containing amino acids caused by non-thermal dielectric-barrier discharge atmospheric-pressure plasma.

Since non-thermal atmospheric-pressure ("cold") plasma sources, such as the dielectric-barrier discharge (DBD), have appeared to be remarkably active in wound healing medicine, the elucidation of cold plasma safety and possible secondary undesirable effects becomes of paramount importance. Selenium-containing amino acids, which are commonly incorporated in many enzymes, came in the spotlight for elucidating the plasma impact as easily oxidizable natural targets. The scope of this study was to analyse the impact of non-thermal plasma on selenium-containing amino acids. Moreover, this research examines the emerging role of metals in the context of oxidation potency of reactive species generated by plasma. The dielectric barrier discharge (DBD) was used to treat 4 mM solutions of Se-(methyl)seleno-l-cysteine (1), l-selenomethionine (2) and seleno-l-cystine (3) for varying treatment times to investigate possible degradation products. In this study we used two redox active iron complexes as well as a redox inert zinc complex in order to compare their capacity to affect chemical modifications caused by plasma. The solutions with selenium-containing amino acids after plasma treatment were analyzed by IR spectroscopy, electrospray ionization mass spectrometry (ESI-MS) and High Performance Liquid Chromatography (HPLC). Several oxidation products were observed as a consequence of plasma treatment, namely: Se-(methyl)seleno-l-cysteine (1) and l-selenomethionine (2) were oxidized to selenoxide and selendioxide derivatives, wheres the Se-Se dimer, seleno-l-cystine (3), was converted to Se-cysteine and seleninic acid. Additionally and to our surprise, redox active iron(ii) and iron(iii) complexes as well as the non-redox active zinc(ii) complex caused the same oxidation pattern when added to the plasma treatment mixtures. Finally, a comparison of the results from Se-containing amino acids with those of their S-containing counterparts revealed that Se-containing amino acids are less prone to cold plasma oxidation than S-containing molecules. By elucidating molecular details of plasma-biomolecule interactions herein we aim to contribute to a better understanding of the complex beneficial medical effects of cold plasma treatments.

Effect of Ni-Doping on the Optical, Structural, and Electrochemical Properties of Ag29Nanoclusters.

Atomically precise metal nanoclusters (NCs) can be compositionally controlled at the single-atom level, but understanding structure-property correlations is required for tailoring specific optical properties. Here, the impact of Ni atom doping on the optical, structural, and electrochemical properties of atomically precise 1,3-benzene dithiol (BDT) protected Ag29 NCs is studied. The Ni-doped Ag29 (NiAg28(BDT)12) NCs, are synthesized using a co-reduction method and characterized using electrospray ionization mass spectrometry (ESI MS), ion mobility spectrometry (IMS), and X-ray photoelectron spectroscopy (XPS). Only a single Ni atom doping can be achieved despite changing the precursor concentration. Ni doping in Ag29 NCs exhibits enhanced thermal stability, and electrocatalytic oxygen evolution reaction (OER) compared to the parent NCs. Density functional theory (DFT) calculations predict the geometry and optical properties of the parent and NiAg28(BDT)12 NCs. DFT is also used to study the systematic single-atom doping effect of metals such as Au, Cu, and Pt into Ag29 NCs and suggests that with Ni and Pt, the d atomic orbitals contribute to creating superatomic orbitals, which is not seen with other dopants or the parent cluster. The emission mechanism is dominated by a charge transfer from the ligands into the Ag core cluster regardless of the dopant.

Rhamnolipid-Enriched PA3 Fraction from Pseudomonas aeruginosa SWUC02 Primes Chili Plant Defense Against Anthracnose

Chili anthracnose, caused by Colletotrichum truncatum, causes significant yield loss in chili production. In this study, we investigated the elicitor properties of a rhamnolipid (RL)-enriched PA3 fraction derived from Pseudomonas aeruginosa SWUC02 in inducing systemic resistance in yellow chili seedlings and antifungal activity against C. truncatum CFPL01 (Col). Fractionation of the ethyl acetate extract yielded 12 fractions, with PA3 demonstrating the most effective disease suppression, reducing the disease severity index to 4 ± 7.35% at 7 days post-inoculation compared with Col inoculation alone (83 ± 23.57%). PA3 also exhibited direct antifungal activity, inhibiting Col mycelial growth by 41 ± 0.96% at 200 µg/mL. Subfractionation revealed PA3 as a mixture of mono- and di-RLs, confirmed by 1H nuclear magnetic resonance and electrospray ionization mass spectrometry data. Additionally, PA3 enhanced seed germination and promoted plant growth without causing phytotoxicity. Transcriptomics revealed that PA3 pre-treatment prior to Col infection primed the defense response, upregulating defense-related genes involved in the phenylpropanoid, flavonoid, and jasmonic acid biosynthesis pathways, as well as those associated with cell wall reinforcement. Our findings highlight the potential of RL-enriched PA3 as both an antifungal agent and a plant defense elicitor, with transcriptome data providing new insights into defense priming and resistance pathways in chili, offering an eco-friendly solution for sustainable anthracnose management.

Improved ion detection sensitivity in mass spectrometry imaging using tapping-mode scanning probe electrospray ionization to visualize localized lipids in mouse testes.

Mass spectrometry imaging (MSI) is a promising analytical method to visualize the distribution of lipids in biological tissues. To clarify the relationship between cellular distribution and lipid types in a tissue, it is crucial to achieve both an improvement in ion detection sensitivity and a reduction in the ionization area. We report methods for improving the efficiency of ion transfer to a mass spectrometer and miniaturizing the extraction area of a sample for tapping-mode scanning probe electrospray ionization (t-SPESI), atmospheric pressure sampling, and ionization methods. To verify the efficacy of the new t-SPESI measurement system, MSI was performed on mouse testes with a pixel size of 5µm. Lipid images of the testes from wild-type (WT) and lysophospholipid acyltransferase 3 (LPLAT3) knockout mice revealed the characteristic distribution of docosahexaenoic acid-containing phospholipids (DHA-PLs). A comparison of the ion images obtained by MSI and optical images of the same tissues stained with hematoxylin and eosin suggested that the distribution of DHA-PLs was significantly altered by spermatogenesis in the WT mouse testes.

Determination of sacubitril and seven sartan drugs in serum samples by online solid phase extraction-liquid chromatography-tandem mass spectrometry

An online solid phase extraction–ultra performance liquid chromatography–tandem mass spectrometry detection method was developed for the simultaneous determination of sacubitril and seven sartan drugs in blood serum in this study. The compounds were separated through a C18 column. Mass spectrometry of the samples was performed using a jet stream electrospray ion source (AJS, ESI+). The samples were detected via a multiple reaction monitoring (MRM) mode and quantified via a stable isotope internal standard method. 900 μL of prepared sample was injected and a run time of 12.5 minutes was obtained in this proposed method. The eight examined target compounds showed good linearity (r2＞0.994) in the corresponding mass concentration range and a lower limit of quantitation (LLOQ) was in the range of 0.05–0.1 μg/L. The recovery of the spiked serum samples ranged from 90.90 % to 106.20 % with relative standard deviations (RSDs) of 4.57 %–9.27 %. Five target compounds were detected in the actual serum samples using this method. The proposed method is simple to use, sensitive, accurate, and suitable for the trace detection of sacubitril and seven sartan drugs present in serum samples. The method can meet the needs for clinical monitoring of blood concentrations of this type of drug, while providing detection technology support for the development of compound preparations of sacubitril and other sartan drugs.

Structure and Stabilities of Solution and Gas Phase Protein Complexes.

Collision-induced unfolding (CIU) has provided new levels of understanding of the stabilities and structure(s) for gas phase protein and protein complex ions formed by electrospray ionization (ESI). Variable-temperature (vT-ESI) data provide complementary information about temperature-induced folding/unfolding (TIU) reactions of solution phase ions. Results obtained by using CIU and TIU provide complementary information about stabilities of gas phase versus solution phase ions. Such comparisons may provide the most direct experimental approach to answer a long-standing question from Fred McLafferty: "For how long, under what conditions, and to what extent, can solution structure be retained without solvent?" Answers to this question require greater understanding of the (i) structure(s), stabilities, and dynamics of proteins/protein complexes in solution prior to ESI; (ii) effects of water removal by droplet fission and "freeze-drying" by evaporation of water from the nanodroplet; and (iii) potential reactions and structural changes that may occur as the ions traverse the heated capillary, the final stage in the transition to solvent-free gas phase ions. Here, we employ vT-ESI coupled with ion mobility-mass spectrometry (IM-MS) as a means to provide more detailed answers to the above-mentioned questions. Apo- and metalated-metallothionein-2A (MT), a cysteine-rich metal binding protein, and various proteoforms of transthyretin (TTR), a homotetrameric (56 kDa) retinol and thyroxine transporter protein complex were studied to examine distinct features of CIU and TIU across two different types of protein complexes. The results in this work shed light on the capabilities of CIU, TIU, and average charge state (Zavg) for probing the rugged energy landscape of native proteins and highlights the effects of water and cofactors (metal ions) on the structure and stabilities of proteins and protein complexes.

Application of UHPLC-ESI-MS/MS to Identify Free Radicals via Spin Trapping with BMPO.

Free radicals play an important role in many chemical and biological processes, but due to their highly reactive and short-lived nature, they evade most analytical techniques, limiting our understanding of their formation and reactivity. Spin trapping molecules can react with free radicals to form radical adducts with lifetimes long enough for analysis. Mass spectrometry is an attractive way to identify radical adducts, but due to their radical nature, they form untraditional oxidized [M]+ and reduced [M+2H]+ ions, which complicates the interpretation of mass spectrometry analysis. This work uses simplified mixtures of radicals generated in both water and dimethyl sulfoxide (DMSO) with spin trap 5-tert-butoxycarbonyl-5-methyl-1-pyrroline-N-oxide (BMPO), to elucidate the behavior of nitroxide spin traps in electrospray ionization (ESI) mass spectrometry (MS) interfaced with liquid chromatography (LC). This study proposes a disproportionation mechanism to explain the formation of the oxidized and reduced BMPO adducts detected by LC-ESI-MS and explores the formation of "di-adducts" through radical recombination. We finally present a framework for differentiating between the different types of ions using collision induced fragmentation mass spectra (MS/MS). This work offers a comprehensive investigation into the behavior of radical adducts in ESI-MS to streamline the identification of organic radicals and advance understanding of radical chemistry.

Discovery of two new long chain fatty acid esters of flavonol glycoside from Pollen Typhae Carbonisatus and their protective effects on HBMECs

ABATRACT Two new compounds, namely, isorhamnenin-3-O-neohesperidin-6′′-linoleic acid ester (1) and isorhamnenin-3-O-neohesperidin-6′′-palmitate (2), along with 17 known compounds, were isolated from Pollen Typhae Carbonisatus (PTC). Their structures were elucidated on the basis of one-dimensional (1D)-, two-dimensional (2D)-nuclear magnetic resonance (NMR), and high-resolution electrospray ionization mass spectrometry (HRESIMS) spectroscopic data analyses. The two new compounds (1 and 2) exhibited a protective effect in a dose-dependent manner and promoted tube generation in the oxygen-glucose deprivation/reoxygenation (OGD/R)-induced human brain microvascular endothelial cells (HBMECs).

Determination of perfluorooctanoic acid and perfluorooctane sulphonic acid in edible vegetable oils using QuEChERS and high performance liquid chromatography-tandem mass spectrometry

ABSTRACT In this study, a rapid, effective method based on QuEChERS was optimised and validated for determination of perfluorooctanoic acid (PFOA) and perfluorooctane sulphonic acid (PFOS) in edible vegetable oils by high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS). Samples were simply vortex extracted with methanol containing 1% ammonia solution and purified through C18, PSA and GCB, followed by determination with HPLC-MS/MS. The detection parameters, including the mobile phase composition, the chromatographic column, as well as the sample preparation conditions, such as the choice of extraction solvents and the dosage of C18, PSA and GCB cleanup agents were optimised. The quantification of target compounds was carried out using stable isotope internal standards to compensate for any matrix effect during the electrospray ionisation process in the mass spectrometry. The developed method has demonstrated commendable accuracy, with recovery percentages ranging from 93.76% to 108.96%, and exhibits excellent precision, as indicated by relative standard deviation not exceeding 20%. Furthermore, the method has been validated for its selectivity, linearity of response, and sensitivity. The proposed method is beneficial for gathering residue data on PFOA and PFOS, allowing for the evaluation of their contamination levels in edible vegetable oils available in the Chinese market and providing scientific backing for regulatory supervision.

Paralytic Shellfish Poisoning (PSP) Toxins in Bivalve Molluscs from Southern Italy Analysed by Liquid Chromatography Coupled with High-Resolution Mass Spectrometry (UHPLC-HRMS/MS).

A new method for simultaneous determination by liquid chromatography coupled with high resolution mass spectrometry (UHPLC-HRMS/MS) of 14 paralytic shellfish poisoning toxins (PSP), that is, Saxitoxin, Neosaxitoxin, Gonyautoxins and their respective variants, in bivalve molluscs, is herein described. The samples were extracted by acetic acid solution, then analysed by UHPLC coupled with a Q-Exactive Orbitrap Plus high resolution mass spectrometer, by electrospray ionization mode (ESI) with no further clean up step. The analysis was carried out by monitoring both the exact mass of the molecular precursor ion of each compound (in mass scan mode, resolution at 70,000 FWHM) and its respective fragmentation patterns (two product ions) with mass accuracy greater than 5 ppm. The analytical performance of the method was evaluated calculating trueness, as mean recoveries of each biotoxin, between 77.8% and 111.9%, a within-laboratory reproducibility (RSDR) between 3.6% and 12.2%, the specificity, the linearity of detector response, and the ruggedness for slight changes The results of the validation study demonstrate this method fits for the purposes of the official control of PSP toxins in molluscs. The results of two years of monitoring in local mussel farms are also reported, showing that no significant concerns for food safety in the monitored productions.

Rapid Exploration of Chemical Space by High-Throughput Desorption Electrospray Ionization Mass Spectrometry.

This study leverages accelerated reactions at the solution/air interface of microdroplets generated by desorption electrospray ionization (DESI) to explore the chemical space. DESI is utilized to synthesize drug analogs at an overall rate of 1 reaction mixture per second, working on the low-nanogram scale. Transformations of multiple drug molecules at specific functionalities (phenol, hydroxyl, amino, carbonyl, phenyl, thiophenyl, and alkenyl) are achieved using electrophilic/nucleophilic, redox, C-H functionalization, and coupling reactions. These transformations occur under ambient conditions on the millisecond time scale with direct detection of products being successful in all but three of the reaction types studied. The large scope (22 bioactive compounds, >20 chemical transformations, and >300 functionalization reagents) and high speed (>3000 reactions/hour) provide access to a wide array of drug analogs that can be used for bioactivity testing. A total of ∼6800 unique reactions were examined through a data-driven workflow, and more than 3000 unique derivatives (∼44%) were identified tentatively by the m/z value and signal-to-control ratio in single-stage mass spectrometry (MS) analysis, with over 1000 being further characterized by tandem MS. The speed of the DESI-MS reaction screen provides potential advantages for emerging machine learning-based predictions of organic synthesis, and it sets the stage for future online DESI-MS bioassays and scaled-up microdroplet synthesis before formal characterization of hit compounds is sought using traditional methods of drug discovery.

Accelerated Amyloid Aggregation Dynamics of Intrinsically Disordered Proteins in Heavy Water.

We explored the influence of D2O on the fibrillation kinetics and structural dynamics of amyloid intrinsically disordered proteins (IDPs), including α-synuclein, amyloid-β 1-42, and K18. Our findings revealed that fibrillation of IDPs was accelerated in D2O compared to that in H2O, exhibiting faster kinetics in contrast to the structured protein, insulin. Structural investigations using electrospray ionization ion mobility mass spectrometry and small-angle X-ray scattering combined with molecular dynamics simulations demonstrated that IDPs did not show significant structural changes that could influence accelerated fibrillation in D2O. Umbrella sampling of protein protofibrils verified that an increased level of hydrogen bonding of D2O and enhanced hydrophobic interactions stabilized β-sheet structured fibrils in D2O. These findings indicate that stabilizing β-sheet fibrils and a more hydrophobic microenvironment in D2O result in enhanced and faster fibrillation of IDPs. The study highlights the importance of considering D2O's differential impact on protein interactions when conducting structural and kinetic analyses, particularly for native peptides and proteins.

Valorisation of Winery By-Products: Revealing the Polyphenolic Profile of Grape Stems and Their Inhibitory Effects on Skin Aging-Enzymes for Cosmetic and Pharmaceutical Applications.

Grape (Vitis vinifera L.) stems, a by-product of winemaking, possess significant potential value due to their rich polyphenolic composition, which allows their exploitation for cosmetic and pharmaceutical applications. This presents a promising opportunity for valorisation aimed at developing innovative products with potential health-promoting effects. In this study, the polyphenolic profile of extracts from grape stems of seven white grape varieties was determined using spectrophotometric and chromatographic methods, specifically high-performance liquid chromatography coupled with a photodiode array detector and electrospray ionization multi-stage mass spectrometry (HPLC-PDA-ESI-MSn), as well as on their ferric-reducing antioxidant power (FRAP) and radical scavenging capacity, using 2,2-diphenyl-1-picrylhydrazyl (DPPH●) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS●+) radicals. This study also evaluated the anti-aging activity and skin depigmenting activity of these extracts. These findings revealed a diverse polyphenolic profile, encompassing proanthocyanidins and catechin derivatives (PCDs), phenolic acids, and flavonols. Among the varieties studied, 'Códega do Larinho' exhibited the highest concentrations of six distinct polyphenols and the highest total phenolic content. It also demonstrated the highest results for antioxidant capacity and elastase and tyrosinase inhibition. Pearson's correlation analysis showed a significant positive correlation between certain PCDs with both FRAP and DPPH assays, as well as between the identified flavonols and anti-elastase activity. These results underscore the potential health benefits of grape stem extracts and emphasize the importance of their polyphenolic composition in enhancing antioxidant and anti-aging properties, thus supporting their application in different industries.

Atomically Precise Rhodium‐Gold Carbonyl Nanoclusters: In‐Depth Synthesis and Multivalence Investigation of [Rh16Au6(CO)36]6−, and its Correlation with [Rh10Au(CO)26]3−

AbstractThe synthesis of the previously‐reported atomically‐precise [Rh16Au6(CO)36]6− (1) nanocluster has been optimized by screening different reaction variables like solvent, stoichiometric ratio, counterion and atmosphere. Its multivalence properties have also been studied by means of electrochemical and spectroelectrochemical techniques; the results pointed out a remarkable electron‐sponge behaviour, as under such experimental conditions 1 is capable to release one electron and accept up to four more, while retaining its molecular structure. This scarce structural variation has been confirmed through DFT calculations on those species whose redox processes showed the maximum reversibility, namely [Rh16Au6(CO)36]7− and [Rh16Au6(CO)36]5−. Moreover, we report herein the isolation and characterization of the new [Rh10Au(CO)26]3− (2) nanocluster, which represents one of the major fallouts of the synthetic pathway investigation of 1. Finally, we disclosed the existence of further species that, from preliminary results, appear to be new large atomically‐precise Rh−Au nanoclusters, which will be the subject of future investigations. Cluster 2 has been identified via single‐crystal X‐ray diffraction analysis, and the characterization of both clusters 1 and 2 completed by infrared (IR) spectroscopy and Electrospray Ionization Mass Spectrometry (ESI‐MS).

Parallel Analyses by Mass Spectrometry (MS) and Reverse Phase Protein Array (RPPA) Reveal Complementary Proteomic Profiles in Triple-Negative Breast Cancer (TNBC) Patient Tissues and Cell Cultures.

High-plex proteomic technologies have made substantial contributions to mechanism studies and biomarker discovery in complex diseases, particularly cancer. Despite technological advancements, inherent limitations in individual proteomic approaches persist, impeding the achievement of comprehensive quantitative insights into the proteome. In this study, we employed two widely used proteomic technologies, mass spectrometry (MS) and reverse phase protein array (RPPA) to analyze identical samples, aiming to systematically assess the outcomes and performance of the different technologies. Additionally, we sought to establish an integrated workflow by combining these two proteomic approaches to augment the coverage of protein targets for discovery purposes. We used 14 fresh frozen tissue samples from triple-negative breast cancer (TNBC: seven tumors versus seven adjacent non-cancerous tissues) and cell line samples to evaluate both technologies and implement this dual-proteomic strategy. Using a single-step protein denaturation and extraction protocol, protein samples were subjected to reverse-phase liquid chromatography (LC) followed by electrospray ionization (ESI)-mediated MS/MS for proteomic profiling. Concurrently, identical sample aliquots were analyzed by RPPA for profiling of over 300 proteins and phosphoproteins that are in key signaling pathways or druggable targets in cancer. Both proteomic methods demonstrated the expected ability to differentiate samples by groups, revealing distinct proteomic patterns under various experimental conditions, albeit with minimal overlap in identified targets. Mechanism-based analysis uncovered divergent biological processes identified with the two proteomic technologies, capitalizing on their complementary exploratory potential.

Lignocellulose biodegradation to humic substances in cow manure-straw composting: Characterization of dissolved organic matter and microbial community succession

Composting, a sustainable practice, facilitates the biodegradation of organic waste, notably lignocellulosic biomass, into value-added humic substances. Despite its potential, the application of electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI FT-ICR MS) to characterize dissolved organic matter (DOM) for assessing the changes in maturity during cow manure-straw composting is underexplored. Furthermore, the link between these changes, microbial community succession, and the biochemical pathways of humus formation is seldom investigated. This study leveraged ESI FT-ICR MS and metagenomic analysis to elucidate the molecular changes in DOM, identified key microbes in humus formation, and traced the humus formation pathway during composting. The results highlighted the crucial role of microorganisms such as Thermobifida, Luteimonas, Ascomycota, and Chloroflexi in accelerating the breakdown and transformation of plant biopolymers. Large molecular nitrogen compounds from cow manure-straw were converted into unsaturated, aromatic oxygen compounds, which resemble humic substances in their chemical properties. The ESI FT-ICR MS data revealed that humus formation occurred through a series of reactions, including protein deamination, lignin delignification, and decarbonylation. This research offered new light on strategies to enhance the stabilization and humification of cow manure-straw composting, contributing to more effective composting processes.

Exploring peptide dendrimers for intestinal lymphatic targeting: formulation and evaluation of peptide dendrimer conjugated liposomes for enhancing the oral bioavailability of Asenapine maleate

Asenapine maleate (ASPM) is a second-generation atypical antipsychotic that is approved for treating acute schizophrenia and bipolar disorder in adults by the US FDA. The major downside of ASPM therapy is rapid, extensive first-pass hepatic metabolism following its oral administration with a very low oral bioavailability of < 2%. In this work, we developed ASPM nanoformulations conjugated with ligands such as arginine-glycine-aspartic acid (RGD) and peptide dendrimers (PDs) with the intention of improving the oral bioavailability of the drug by targeting it to the intestinal lymphatic system (ILS). Peptide dendrimers (PDs), both lipidated and nonlipidated, were synthesized by Fmoc solid phase peptide synthesis (SPPS). Reverse phase high performance chromatography (RP-HPLC) was used to purify the synthesized PDs, and the PDs were characterized by differential scanning calorimetry (DSC) electrospray ionization mass spectroscopy (ESI+-MS), Nuclear magnetic resonance (NMR) and Fourier transform infrared (FTIR) spectroscopy. The thin film hydration method was used to prepare liposomes, and the process variables affecting the liposome parameters were optimized using the Box‒Behnken design (BBD).Liposomes were PEGylated using DSPE-PEG-COOH2000 and further conjugated with ligands (RGD, PD-1 and PD-2) using EDC-NHS chemistry. The formulation was characterized using different spectroscopic techniques. In vitro, cell line studies, such as cytotoxicity, cell uptake, uptake mechanism, and receptor saturation studies, were performed on both Caco2 and Raji-B cells. The pharmacokinetic parameters of the developed liposomal formulation were evaluated using pharmacokinetic studies on Sprague- Dawley (SD) rats. The psychostimulant-induced hyperactivity model was used to evaluate the pharmacodynamic performance of the developed formulations by measuring the reversal of hyperlocomotor activity induced by levodopa-carbidopa.

Advancing Targeted Metabolomics Using Cyanopropyl-Based Liquid Chromatography Tandem Mass Spectrometry.

The change of metabolic pathways is recognized as the key to disease discovery prompting the development of ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS)-based quantitative platforms to explore the dynamic metabolite profiles of organisms. In this study, a liquid chromatography method based on cyanopropyl (CN) was developed. By adjusting the pH environment of the column, we achieved the elution of 51 metabolites spanning the most comprehensive set of biological pathways currently known. Offering rapid chromatography, efficient separation, and green chemistry benefits, the method encompasses nucleosides and nucleotides, the oxidative-redox metabolome, the glycolysis pathway, the pentose phosphate pathway, the purine de novo pathway, amino acids, and neurological disorder-related metabolites. The mass spectrometry was equipped with electrospray ionization in both positive and negative modes with scheduled multiple reactions monitoring. The validation of the method involved a comprehensive assessment of linearity, accuracy, precision, and matrix effect. The linear range was from 1.0 to 2000 ng mL-1 with a high correlation coefficient (r > 0.99). The LOD ranged from 0.1 to 10 ng mL-1, and the LOQ ranged from 0.1 to 25 ng mL-1. The overall recovery ranged from 81.3% to 117.8%, with RSD < 15.1%. Subsequently, an analysis of metabolites was conducted in dSH-SY5Y neuroblastoma cells with 6-hydroxydopamine, a commonly used neurotoxin in neurodegenerative diseases. The results demonstrate that neurotoxin-induced mitochondrial damage significantly altered related analytes, corroborating previous estimates and validating the feasibility and reliability of the bioanalytical platform.