Search Results (1,016)

Peptide mapping is a well-established method for confirming the identity of therapeutic proteins as part of batch release testing and product characterization for regulatory filings. Traditionally based on enzymatic digestion followed by reversed-phase liquid chromatography and UV detection, the method has evolved with technological advancements to incorporate mass spectrometry (MS), enabling more detailed structural insights. Residue-level confirmation of amino acid sequences requires MS/MS fragmentation, which produces large amounts of data that must be processed using specialized software. In regulated environments, the use of academic algorithms is often limited by validation requirements, making it necessary to rely on commercially approved tools, although their built-in scoring systems have limitations that can affect sequence assignment accuracy. Here, we present representative examples of incorrect peptide assignments generated by commercial software. In antibody sequence analysis, misidentifications resulted from isobaric and near-isobaric dipeptides (e.g., SA vs. GT). Additional examples from the analysis of SARS-CoV-2 spike protein variants revealed software-induced artifacts, including artificial succinylation of aspartic acid residues to compensate for sequence mismatches, and incorrect deamidation site assignments due to misinterpretation of isotopic peaks. These findings underscore the necessity for expert manual review of MS/MS data, even when using validated commercial platforms, and highlight the molecular challenges in distinguishing true sequence variants from software-driven artifacts. Full article

This study presents nanofractionation analytics coupled with in vivo profiling of zebrafish embryo paralysis and lethality in response to toxins in cone snail venoms. The focus of this study is on the development of this approach using venoms of Conus marmoreus, Conus ebraeus, and Conus bandanus. In brief, cone snail venoms were separated using reversed-phase chromatography following high-resolution nanofractionation on microplates with parallel mass spectrometry, enabled via a post-column flow split. All collected fractions were dried overnight, followed by assays on zebrafish embryos. For the paralysis assessment, we monitored swimming behavior and swimming distance and found that exposure to cone snail toxins led to paralysis and decreased movement and swim distance. To correlate the masses of eluted toxins with their paralyzing effects and potency, we compared the fractionation retention time versus normalized swimming distance. This allowed identification of the masses of toxins with paralyzing bioactivity, which were predominantly conopeptides. To assess lethality, zebrafish embryos were exposed to fractionated toxins for 24 h, after which they were inspected. The lethal doses and correlated toxins were identified by comparing retention times of fractionation versus the lethal dose values calculated for each fraction. We found that the most lethal venom was from C. bandanus, displaying the largest number of lethal peptides, followed by C. marmoreus and C. ebraeus. On the other hand, the most paralytic venom was from C. ebraeus, presenting a higher number of peptides with non-lethal paralytic effects, followed by C. bandanus and C. marmoreus. This study provides a pipeline to rapidly identify paralytic and lethal cone snail venom toxins using the zebrafish embryo model. Full article

The sustainable revalorization of porcine blood is crucial due to the large volumes daily generated in slaughterhouses. The aim of this study was to obtain a novel ingredient rich in free amino acids and bioactive peptides from the sequential hydrolysis of porcine blood. Porcine blood was hydrolyzed with Alcalase 4.0 L and Protana™ Prime enzymes, followed by molecular weight fractionation (<10 kDa) and spray-drying. The antioxidant, hypoglycemic, and anti-inflammatory bioactivities of the resulting hydrolysate (PBSH) were studied in vitro. Further fractionation by reversed-phase high-performance liquid chromatography (RP-HPLC) was performed to isolate the most bioactive fraction based on polarity. Peptides from fraction 1 (F1) were identified using LC-MS/MS and analyzed in silico. Finally, some peptides were synthesized, and their bioactivity was subsequently assessed. PBSH hydrolysate showed antioxidant activity with IC₅₀ values of 2.09, 135.05, and 26.73 mg/mL for ABTS, FRAP, and DPPH assays, respectively. Additionally, PBSH exhibited hypoglycemic, anti-inflammatory, and immunomodulatory potential through the inhibition of DPP-IV (82.78%), NEP (84.72%), TACE (50.79%), and MGL (69.08%) enzymes at a concentration of 20, 20, 100, and 20 mg/mL, respectively. Peptides PDDFNPS, FPPKPKD, DNPIPK, GHLDDLPG, and GDL were identified in the most polar and bioactive fraction (F1) and proved a synergistic hypoglycemic effect at a concentration of 1 mmol/L. The peptide PDDFNPS exhibited multifunctional properties with 56.43% inhibition of DPP-IV and 83.54% inhibition of NEP. PBSH resulted in a novel functional ingredient for animal feed as it contains a variety of identified bioactive peptides and a high amount of free amino acids. Full article

Young child formula (YCF) products are important sources of nutrients for children 1–3 years of age. Salt equilibria and protein glycation are two of the crucial aspects affecting nutritional properties and digestive behaviors of YCF, but detailed insights into these two aspects of YCF products remains limited. This study analyzed the distribution of salts and the level of protein glycation in 25 commercial YCF products from the retail market in China. The YCF products were reconstituted (12 g of powder per 100 g of water) and the distribution of calcium and phosphorus between the sedimentable (at 200× g), protein-associated and soluble (10 kDa-permeable) fractions were determined. Blocked lysine and 5-hydroxymethylfurfural were analyzed using reversed-phase high-performance liquid chromatography. Varying proportions of calcium (3.0–39.3%) and phosphorus (1.2–29.8%) were sedimentable for the products. Notable proportions of calcium (28.9–62.7%) and phosphorus (27.4–57.9%) were associated with the proteins. The remainder of the calcium (24.9–41.4%) and phosphorus (34.2–62.1%) were soluble. When expressing the protein-associated calcium as a function of casein, i.e., casein mineralization, large differences (~1.7 fold) were found among products. Variation in blocked lysine (7.4–19.2% of total lysine) and 5-hydroxymethylfurfural contents (3.0–7.0 mg/100 g protein) among products was also observed, suggesting notable differences in heat-load during processing. This study revealed notable variation in salt distribution and protein glycation among the YCF products. These findings underscore the critical need for manufacturers to optimize formulation and processing approaches, e.g., using milk with a low level of casein mineralization and using milk protein sources as concentrated liquid rather than powder to reduce protein glycation, to improve nutritional properties of the products. Full article

Searching anode materials is an important task for the development of sodium-ion batteries. In this regard, bronze-phase titanium dioxide, TiO₂(B), has been considered as one of the promising materials, owing to its crystal structure with open channels and voids facilitating Na⁺ diffusion and storage. However, the electrochemical de-/sodiation mechanism of TiO₂(B) has not been clearly comprehended, and further experiments are required. Herein, in situ and ex situ observations by a combination of X-ray photoelectron spectroscopy, X-ray diffraction, Raman spectroscopy, gas chromatography–mass spectrometry was used to provide additional insights into the electrochemical reaction scenario of bronze-phase TiO₂ in Na-ion batteries. The findings reveal that de-/sodiation of TiO₂(B) occurs through a reversible intercalation reaction and without the involvement of the conversion reaction (no metallic titanium is formed and no oxygen is released). At the same time, upon the first Na⁺ uptake process, crystalline TiO₂(B) becomes partially amorphous, but is still driven by the Ti⁴⁺/Ti³⁺ redox couple. Importantly, TiO₂(B) has pseudocapacitive electrochemical behavior during de-/sodiation based on a quantitative analysis of the cyclic voltammetry data. The results obtained in this study complement existing insights into the sodium storage mechanisms of TiO₂(B) and provide useful knowledge for further improving its anode performance for SIBs application. Full article

Lunasin is a peptide found in the soybean albumin 2S subunit, which has important bioactivities, such as anticancer and antioxidant. Recently, peptides similar to soybean lunasin have been reported in other cereal and legume seeds; for this reason, it is considered important to carry out a review that compiles this information, whose interest lies mainly in the bioactive properties of these peptides. The peptides reported in the literature contained in barley, wheat, rye, triticale, oat, black nightshade, amaranth, bean, chickpea, grass pea, lentil, and pea are analyzed and described. Isolation methods such as ion exchange chromatography, immunoaffinity column chromatography, Western blot, reversed-phase chromatography coupled to an electrospray ionization source, extraction with water and dialysis, and extraction with PBS, and tests such as internalization, radical scavenging, chelating, cytotoxicity in cancer cell lines essays, and histone acetyltransferase inhibition essays were carried out to identify their anticancer properties. It is worth mentioning that the in silico analyses of proteins in which the lunasin-like peptide is located have been developed in some of these seeds; however, more studies are needed in order to confirm sequence similarity to that of the lunasin peptide. Further work is needed in order to identify the sequence of these lunasin-like peptides and corroborate their similarity to that of the lunasin, such as the development of specific antibodies for each lunasin-like peptide reported in each type of seeds. This document aims to compile the advances in the research on lunasin-like peptides and their bioactivities to have a better understanding of the current advances related to these peptides. Full article

Geum aleppicum Jacq. (yellow avens), a species traditionally used in folk medicine, remains understudied in the ethnopharmacological aspects. In this study, we comprehensively evaluated the phytochemical composition and biological activity of a hydroethanolic (50:50, v/v) extract from the aerial parts of G. aleppicum collected in Kazakhstan. Using the high-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (HPLC-ESI-QTOF-MS/MS), we identified 24 compounds, predominantly phenolic acids, flavonoids, tannins, and triterpenoids. The major compound was ellagic acid (2.28 mg/g dry extract) as revealed by the reverse phase high-performance liquid chromatography–diode array detector (RP-HPLC-DAD). The extract exhibited a high polyphenol content (131.45 mg GAE/g) and strong antioxidant activity in Ferric Reducing Antioxidant Power (FRAP) assay and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay (3.82 ± 0.07 mmol Fe²⁺/g and 106.61 ± 0.89 mg GAE/g, respectively). Antimicrobial assay of the extract revealed notable antifungal activity against Candida spp., especially against C. glabrata and C. tropicalis with minimum inhibitory concentration (MIC) of as low as 0.125 mg/mL, showing fungistatic effect. Although the extract inhibited the cytopathic effect induced by Human Herpesvirus 1 (HHV-1) in VERO cells, it did not significantly reduce viral replication. Moreover, among human cancer cell lines studied, the extract exerted moderate and selective cytotoxicity against A549 lung cancer cells (CC₅₀ = 75.51 µg/mL, SI = 9). These findings highlight G. aleppicum as a rich source of bioactive compounds, especially phenolics, supporting its potential for development of pharmaceutical and cosmetic applications. Full article

Objectives: Ketamine has demonstrated rapid and sustained antidepressant effects; however, its clinical utility is limited by the risk of addiction and systemic side effects. This study aimed to develop ketamine-loaded nanodroplets (Ket-NDs) with high encapsulation efficiency (EE) and stability for targeted low-dose intravenous (IV) administration in a mice model of depression. Low-intensity focused ultrasound (LIFU) was employed to induce transcranial, region-specific drug release in the lateral habenula (LHb). Methods: Ket-NDs were synthesized using a thin-film hydration method with sonication and emulsification, incorporating perfluoropentane as the core material. Characterization was performed using light microscopy, cryogenic scanning electron microscopy (cryo-SEM), transmission electron microscopy, and dynamic light scattering (DLS). Drug EE and loading efficiency (LE) were quantified by reversed-phase high-performance liquid chromatography. A chronic restraint stress model was established, and Ket-NDs were administered intravenously followed by LIFU targeting the LHb. Antidepressant efficacy and biosafety were systematically evaluated. Results: (1) Ket-NDs exhibited uniform spherical morphology and a narrow size distribution, as confirmed by DLS (particle size: 139.75 ± 9.43 nm; Polydispersity index: 0.225 ± 0.025) and cryo-SEM analysis (number-average diameter: 109.5 ± 10.4 nm). The zeta potential was −15.93 ± 5.906 mV, and the formulation remained stable under 4 °C storage. (2) Ket-NDs demonstrated high EE (78.25 ± 16.13%) and LE (15.55 ± 4.49%). (3) In depressive mice, IV administration of Ket-NDs followed by LIFU targeting the LHb significantly improved behavioral outcomes: increased locomotor activity in the open field test, elevated sucrose preference index, and reduced immobility time in the tail suspension test. (4) Safety assessments revealed no significant organ toxicity or brain tissue damage in ultrasound-exposed regions. Conclusions: In summary, this study developed stable Ket-NDs. When combined with LIFU, they enable precise regional drug delivery to the brain, showcasing a promising treatment strategy for depression with reduced systemic side effects. Full article

Background/Objectives: This study aimed to enhance the vaginal permeation of buserelin acetate (BA), a synthetic gonadotropin-releasing hormone (GnRH) analogue, by evaluating various permeation enhancers (PEs) using a validated reversed-phase high-performance liquid chromatography (RP-HPLC) method and an ex vivo porcine vaginal model. Methods: A robust RP-HPLC method was developed and validated according to ICH Q2 (R2) guidelines to enable accurate quantification of BA in permeation samples. The analytical method demonstrated high specificity, linearity (R² = 0.9999), accuracy (98–102%), precision (%RSD < 2%), robustness, and stability. Using this method, ex vivo permeation studies were conducted with six different PEs: 2-hydroxypropyl-β-cyclodextrin, sodium dodecyl sulfate, poloxamer 188, Span 80, Tween 80, and chitosan. Results: Among all tested PEs, chitosan demonstrated the best enhancement of BA permeation. It achieved the highest flux (J) (0.64 ± 0.03 × 10⁻² µg/cm²·h) and apparent permeability coefficient (P_app) (16.20 ± 0.84 × 10⁻⁵ cm/h), both of which were statistically significantly higher (p < 0.05) than those of all other enhancer groups. Kinetic modelling indicated a non-Fickian, biphasic permeation mechanism best described by the Makoid–Banakar model. Conclusions: These findings highlight chitosan’s potential as an effective intravaginal delivery vehicle for peptide therapeutics and establish the validated HPLC method as a reliable platform for future formulation development and translational studies in mucosal drug delivery. Full article

Terephthalic acid (TPA), a major monomer of polyethylene terephthalate (PET), represents a significant challenge in plastic waste management due to its persistence in the environment. In this study, we report a newly developed bacterial consortium capable of using TPA as the sole carbon source in a defined mineral medium. The consortium achieved stationary phase within five days and metabolized approximately 85% of the available TPA. Metabolite analysis by high-performance liquid chromatography (HPLC) and liquid chromatography tandem mass spectrometry (LC-MS/MS) revealed the activation of the benzoate degradation pathway during TPA catabolism. Additionally, the consortium secreted commercially relevant metabolites such as cis,cis-muconic acid and catechol into the culture medium. Genetic profiling using a reverse transcription quantitative polymerase chain reaction (RT-qPCR) and 16S rRNA sequencing identified Paraburkholderia fungorum as the dominant species, suggesting it plays a key role in TPA degradation. The ability of this microbial community to efficiently convert TPA into high-value by-products offers a promising and potentially economically sustainable approach to addressing plastic pollution. Full article

Isorhapontigenin (trans-3,5,4′-trihydroxy-3′-methoxystilbene; ISO), a dietary derivative of resveratrol (trans-3,5,4′-trihydroxystilbene; RES), exhibits diverse health-promoting properties. To facilitate its potential development as a nutraceutical, a simple and reliable high-performance liquid chromatography (HPLC) method was developed and validated for the quantification of ISO in various murine biological matrices. Chromatographic separation was achieved with a reversed-phase HPLC column through a 17 min gradient delivery of a mixture of acetonitrile and formic acid (0.1% v/v) at a flow rate of 1.5 mL/min at 50 °C. Quantification was performed using ultraviolet (UV) detection at 325 nm, with a lower limit of quantification (LLOQ) of 15 ng/mL in both plasma and tissue homogenate samples. The method demonstrated excellent selectivity, accuracy, and precision, and ISO remained stable under the tested conditions. This method was subsequently employed to investigate the tissue distribution of ISO in mice following oral administration at a dose of 200 µmol/kg (equivalent to 51.7 mg/kg). ISO was rapidly absorbed and extensively distributed across major pharmacologically relevant organs. Despite its limited aqueous solubility, its oral absorption was not significantly compromised. Given its oral bioavailability and broad tissue distribution, ISO represents a promising candidate for further nutraceutical development. Full article

Hydrophilic Interaction Liquid Chromatography (HILIC) has emerged as a powerful and versatile analytical technique for the separation and quantification of polar and ionizable compounds, particularly in the field of pharmaceutical impurity profiling. Over the past two decades, HILIC has gained increasing attention due to its compatibility with mass spectrometry, enhanced retention of hydrophilic impurities, and ability to resolve structurally similar degradation products and process-related impurities that are often inadequately retained by reversed-phase chromatography. This comprehensive review presents a critical overview of HILIC-based methodologies applied to impurity profiling in pharmaceutical analysis from early 2005 to the present. Emphasis is placed on the application of HILIC to both small-molecule drugs and large biomolecules. Additionally, the review categorizes analytical configurations into four main groups based on their operational principles and use cases, offering insights into method selection and performance characteristics. This article serves as a valuable resource for researchers and regulatory scientists seeking to apply HILIC in modern impurity profiling and quality control of pharmaceutical products. Full article

Glycolipids are structurally diverse amphiphilic molecules with potential as non-petrochemical-derived bioproducts, including surfactants, emulsifiers, and antioxidants. The different bioactivities associated with this range of glycolipid structures also present opportunities for dietary supplements, cosmetics, and pharmaceuticals. Marine glycolipids are underexplored due to challenges with purification and structural characterisation. Analytical approaches enabling efficient sample purification, isolation, and identification of target glycolipids are crucial to determining the bioactivity and functions of organisms such as shellfish and seaweed. This review summarises advances in analytical methods applicable to marine glycolipids, including extraction and enrichment methods tailored to specific subclasses. Thin-layer chromatography (TLC)-based rapid detection techniques developed for specific subclasses in complex biological samples are discussed, alongside structure identification methods based on liquid chromatography (LC)–electrospray ionisation (ESI)–tandem mass spectrometry (MS/MS). Hydrophilic interaction liquid chromatography (HILIC), reverse-phase liquid chromatography (RPLC), and supercritical fluid chromatography (SFC) coupled with MS detection are reviewed for their application to glycolipids. The application of two-dimensional liquid chromatography (2D-LC) and advanced MS-based approaches that facilitate both the rapid resolution and comprehensive characterisation of molecular species are also reviewed. Full article

Nacre, the iridescent inner layer of mollusk shells, has long been traditionally used in medicine. While we have previously demonstrated its anti-aging effects on muscle and skin, its impact on pancreatic dysfunction and glucose metabolism remains unclear. In this study, we aimed to isolate and identify an active component from nacre extract that improves glucose metabolism and to evaluate its potential to prevent or ameliorate pancreatic dysfunction and glucose metabolic abnormalities in a D-galactose-induced aging mouse model. A polysaccharide component was successfully isolated using a combination of reverse-phase and ion-exchange chromatography. Structural analyses revealed that it was primarily composed of glucose, mannose, and rhamnose, which together accounted for approximately 87% of the total monosaccharide content. Further characterization by FT-IR spectroscopy and MALDI-TOF-MS confirmed its identity as a neutral polysaccharide with glycosidic linkages and an estimated molecular weight of approximately 5000 Da. Intraperitoneal administration of this polysaccharide significantly improved glucose tolerance and prevented a decline in serum insulin levels in D-galactose-induced aging mice. Immunohistochemical analysis of pancreatic tissues revealed that the polysaccharide preserved insulin expression and suppressed the D-galactose-induced upregulation of cellular senescence and apoptosis markers. These findings suggest that this nacre-derived polysaccharide effectively mitigates pancreatic dysfunction and glucose metabolic dysfunction, indicating its potential as a natural therapeutic agent for age-related metabolic disorders. Full article

Background: Compound lipophilicity is a fundamental physicochemical property for determining the pharmacokinetic and pharmacodynamic profiles of therapeutic substances. It is successfully used in the early stages of drug candidates’ design and development. Aim: Taking into account the importance of this parameter, we aimed to assess and compare the utility of a hybrid procedure based on calculation methods and an experimental one for rapid and simple estimation of the lipophilicity of selected neuroleptics such as fluphenazine, triflupromazine, trifluoperazine, flupentixol and zuclopenthixol and their potential new derivatives. Methods: Log P values of the studied compounds were predicted by means of different platforms and algorithms: AlogPs, ilogP, XlogP3, WlogP, MlogP, milogP, logPsilicos-it, logP_consensus, logP_chemaxon and logP_ACD/Labs. The experimental determination of lipophilicity was carried out by reverse-phase thin-layer chromatography (RP-TLC) using three types of stationary phases—RP-2F₂₅₄, RP-8F₂₅₄ and RP-18F₂₅₄—and mobile phases consisted of acetone, acetonitrile and 1,4-dioxane as organic modifiers. Results: Our results provide a confident proposal of optimal chromatographic conditions to experimentally determine the lipophilicity of neuroleptic drugs, including new derivatives. Conclusions: Additionally, for the first time, the paper shows the application of selected topological indices in determining lipophilicity factors and other ADMET parameters of neuroleptics and, in the future, the newly synthesized quinoline derivatives of the studied compounds. Full article