Reversed-phase High-performance Liquid Research Articles

Development and validation of stability-indicating HPLC method for assay of tonabersat in pharmaceutical formulations

A stability-indicating reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed to assay tonabersat and assess its stability in pharmaceutical formulations. Chromatographic separation was achieved using a Kinetex® C18 column (2.6 µm, 150 x 3 mm, 100 Å) at 50 °C, with a 20 µL injection volume. A linear gradient of acetonitrile in water (5 – 33.5 %) was applied for 1 min, followed by a gradual increase to 100 % over 26 min at a flow rate of 0.5 mL/min. Tonabersat and its degradation products were detected at 275 nm and 210 nm, respectively. The optimized method was used to evaluate stability of tonabersat in lipid-based pharmaceutical formulations at 5 ± 3 °C, 25 ± 2°C/60 ± 5 % RH, and 40 ± 2 °C/75 ± 5 % RH over 3 months. The method was validated as per ICH guidelines and demonstrated linearity in the range of 5 – 200 µg/mL (R2 = 0.99994) with good accuracy (98.25 – 101.58 % recovery) and precision (RSD < 2.5 %). The limit of detection and quantitation were 0.8 µg/mL and 5 µg/mL, respectively. Forced degradation studies showed significant degradation under alkaline (90.33 ± 0.80 %), acidic (70.60 ± 1.57 %), and oxidative stress (33.95 ± 0.69 %) at 70 °C, but no degradation was observed under thermal or photolytic stress. No chemical degradation was observed in either formulation on storage. Thus, the method was sensitive, specific, and suitable for stability testing of tonabersat in pharmaceutical formulations.

Validation of Analytical Method for Vitamin A in Bioadhesive Ocular Cationic Nanoemulsion Loaded into Thermosensitive Gel Using RP-HPLC

Various test methods have been previously documented for determining vitamin A levels in different dosage forms. This study specifically examines an isocratic reverse phase-high performance liquid chromatography (RP-HPLC) method designed for the direct extraction of vitamin A. The objective is to validate an analytical method for quantifying vitamin A in bioadhesive cationic nanoemulsions incorporated into thermosensitive gels. The method employs isocratic RP-HPLC with a YMC-Triart C18 column (L1), dimensions of 4.6 mm × 250 nm, particle size of S-5 µm, and a UV detector at λ = 265 nm. The mobile phase consists of HPLC-grade methanol, acetonitrile, and n-hexane in a ratio of 46.5:46.5:7. Validation parameters were assessed including selectivity, linearity, accuracy, precision, limit of quantification (LOQ), and limit of detection (LOD). Correlation coefficients were determined with an R2 value of 0.9995 in the concentration range of 264–396 μg/mL (w/v). Recovery percentages ranged from 99.295% to 99.878%. Repeatability and intermediate precision relative standard deviations (RSD) were found to be 0.318% and 0.254%, respectively. The LOD was established at 2.018 μg/mL, and the LOQ was determined to be 6.114 μg/mL. The results affirm cost-effective and well-suited for the accurate measurement of vitamin A levels in bioadhesive thermosensitive gel formulations.

Novel Green Chromatographic Approaches for Estimation of a Triple Common Cold Pharmaceutical Combination.

Nowadays, there is a strong interest in the scientific community in developing innovative methodologies within a green analytical chemistry framework. Herein, we introduce the first chromatographic approaches for the concurrent estimation of paracetamol (PAR), carbinoxamine (CRX), and pseudoephedrine (PSE) intended to relieve COVID-19 and common cold symptoms. The first method was thin layer chromatography (TLC) densitometry, which depends on the separation of the studied medications on TLC silica gel plates using ethyl acetate: methanol: ammonia (7.0: 3.0: 0.2, by volume) as the developing system, and were scanned at 208.0nm. The data were linear in the ranges of 1-25μg/band for PAR, 1-25μg/band for PSE and 0.1-5μg/band for CRX. The second method was reversed-phase high-performance liquid chromatography separation on a Kromasil C18 column using a mixture of 0.01M phosphate buffer containing 0.1% triethylamine (pH3.5) adjusted with orthophosphoric acid and ethanol at a flow rate of 1.0 mL/min in a gradient program. The separated peaks were detected at 215.0 nm over a concentration range of 10-250μg/mL for PAR, 5-35μg/mL for PSE, and 0.5-25μg/mL for CRX. Both approaches were validated according to International Conference on Harmonization (ICH) guidelines. Finally, the impact of these methods on the environment was evaluated by many tools.

A novel antimicrobial peptide WBp-1 from wheat bran: Purification, characterization and antibacterial potential against Listeria monocytogenes

This study introduces a novel antimicrobial peptide (AMP), WBp-1, isolated from wheat bran and purified via reversed-phase high-performance liquid chromatography. The amino acid sequence, determined as IITGASSGIGKAIAKHFI by LC–MS/MS, was composed predominantly of alkaline and hydrophobic residues. WBp-1 was predicted to be a stable, hydrophobic, cationic peptide with an α-helical structure. Moreover, it displayed significant antibacterial efficacy against Listeria monocytogenes, with a minimum inhibitory concentration of 150 μg/mL. Further mechanistic studies suggest that WBp-1 exerts its bactericidal activity by disrupting cell membrane integrity, impeding peptidoglycan synthesis by binding to penicillin-binding protein 4 via hydrogen bonding, increasing cell permeability, altering membrane potential and fluidity, and altering surface hydrophobicity. Interestingly, WBp-1 showed minimal hemolytic activity and cytotoxicity against LO2 cells, even at 16× MIC. These findings highlight the strong potential of WBp-1 as a novel antibacterial agent and food preservative against Listeria monocytogenes.

Simultaneous and sequential combination of techniques for the sustainable and extensive extraction of proteins and polyphenols from malt rootlets

The emergence of tecnhiques such as microwave-assisted extraction (MAE), enzyme-assisted extraction (EAE), ultrasounds-assisted extraction (UAE), and pressurized liquid extraction (PLE) has brough many benefits but their single use does not guarantee the hollistic extraction of target compounds from complex samples. Taking into account their different principles and that one technique may favour the extraction of certain compounds over others, this work proposes the combination of these techniques for improving extraction yields in the recovery of proteins, as example of large molecule, and phenolic compounds, as example of small molecule, from a recalcitrant sample. No single extraction technique recovered all proteins or phenolic compounds, even after a proper optimization. Among them, PLE was the most advantageous, enabling the extraction of 67 % proteins and 1.20 g gallic acid equivalent (GAE)/100 g sample. Analysis of samples by scanning electron microscopy (SEM) and separation of extracts by reversed-phase high performance liquid chromatography (RP-HPLC) and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) revealed that PLE showed the most distinct efficiency among EAE, MAE, and UAE, whereas the latter three exhibited more similar results. Based in these results, ten different combinations (sequential and/or simultaneous) of these techniques were proposed. PLE in combination with another technique showed a great potential for the extraction of both large and small molecules. Particularly, the combination of EAE and PLE recovered 100 % of proteins and more than three times the phenolic compounds extracted by other methods reported in the literature, even revealing significant synergistic effects.

Anti-biofilm and antibacterial effect of bacteriocin derived from Lactobacillus plantarum on the multidrug-resistant Acinetobacter baumannii

This research examines the impact of bacteriocin derived from Lactobacillus plantarum PTCC 1745 on the biofilm formations of A. baumannii isolates. Bacteriocin derived from L. plantarum PTCC 1745 was obtained through ammonium sulfate precipitation, cation-exchange chromatography, and reversed-phase high-performance liquid chromatography (RP-HPLC). Testing for bacteriocin susceptibility has been conducted using the broth dilution method. The anti-biofilm activity of bacteriocin was evaluated using a microtiter plate method. Quantitative real-time PCR assay evaluated bap gene expression in bacteriocin-treated cells. According to SDS-PAGE, bacteriocin from L. plantarum has a 25-kDa apparent molecular weight. The MICs of bacteriocin ranged from 30 to 120 μg/mL, while the MBCs varied between 60 and 120 μg/mL. Compared to the non-treated group, strains bacteriocin-treated isolates had 59 % less ability to form biofilm. The mean relative expression of the bap gene among the MDR A. baumannii isolates decreased by 52 % compared to the untreated control. This study demonstrated that bacteriocin derived from L. plantarum PTCC 1745 had antibacterial and antibiofilm activity against MDR A. baumannii isolates.

A comprehensive review of analytical strategies for validating RP-HPLC methods of aceclofenac and thiocolchicoside in bulk drug and formulation

This comprehensive review explores the analytical method development and validation of Aceclofenac and Thiocolchicoside bulk drugs and formulations using Reverse Phase High-Performance Liquid Chromatography (RP-HPLC). Aceclofenac, a nonsteroidal anti-inflammatory drug (NSAID), and Thiocolchicoside, a muscle relaxant, are commonly co-formulated for the treatment of pain and inflammation. The review discusses the principles and strategies involved in RP-HPLC method development, emphasizing factors such as stationary phase selection, mobile phase composition, and detection wavelength optimization. Furthermore, it highlights the importance of method validation in pharmaceutical analysis, covering parameters such as specificity, linearity, accuracy, precision, robustness, and system suitability. The literature review section examines previous studies on RP-HPLC methods developed for Aceclofenac and Thiocolchicoside, summarizing key findings and comparing different methodologies. Challenges and limitations encountered during method development and validation are discussed, along with recent advances in RP-HPLC methodology. Finally, the review outlines future research directions and potential applications of RP-HPLC in studying the pharmacokinetics and bioavailability of Aceclofenac and Thiocolchicoside formulations.

Retention factor variation on wide range of mobile phase compositions in reversed-phase high-performance liquid chromatography; a short tutorial

Mobile phase composition remains the major experimental parameter influencing the separation process in reversed-phase high-performance liquid chromatography (RP-HPLC). Its influence on the retention of solutes is explained by the Hildebrand solubility parameter and solvophobic theory, which also considers molecular characteristics of the participants in a separation process. The main empirical dependences (linear and polynomial) between the retention factor and the composition of mobile phase (retention function) are discussed taking into consideration a wide range of mobile phase compositions. Two direct chromatographic indices are resulting from these equations: the extrapolated value of retention factor for zero organic content in mobile phase composition (log kw) and the solvent strength parameter (S), which for a linear retention function is its slope being constant over the entire mobile phase compositions. Another chromatographic index could result from the retention function for zero content of water in mobile phase, but this situation can be applied only for very hydrophobic solutes. For large domains of mobile phase composition, the retention function cannot be described by a single type of dependence. This explains the paradox of generating by extrapolation different logkwvalues for different organic components. For the mobile phase close to pure water the retention function is different from that at average water concentration.

A New Highly Effective Development and Validation of Trastuzumab and Hyaluronidase-Oysk in Bulk and Pharmaceutical Dosage form by Using RP-HPLC Method

A simple, rapid, precise, sensitive, and reproducible Reverse phase High-performance liquid chromatography (RP-HPLC) method has been developed for the quantitative analysis of Trastuzumab and Hyaluronidase-OYSK in the pharmaceutical dosage form. Chromatographic separation of Trastuzumab and Hyaluronidase-OYSK was achieved on Waters Alliance-e2695by using Agilent Eclipse XDB (250x 4.6mm, 5µ) column and the mobile phase containing Acetonitrile: Ammonium formate pH-3.0/OPA in the ratio of 30:70% v/v. The flow rate was 1.0 ml/min; detection was carried out by absorption at 228nm using a photodiode array detector at ambient temperature. The number of theoretical plates and tailing factor for Trastuzumab and Hyaluronidase-OYSK were NLT 2000 and should not be more than 2 respectively. % Relative standard deviation of peak areas of all measurements always less than 2.0. The proposed method was validated according to ICH guidelines. The method was found to be simple, economical, suitable, precise, accurate &amp; robust method for quantitative analysis of Trastuzumab and Hyaluronidase-OYSK study of its stability.

Sorption of 1,2,3,4-Tetrahydroquinoline and Pyridazino[4,5-c]quinoline Derivatives under Conditions of Reversed-Phase High-Performance Liquid Chromatography

Sorption of 1,2,3,4-Tetrahydroquinoline and Pyridazino[4,5-c]quinoline Derivatives under Conditions of Reversed-Phase High-Performance Liquid Chromatography

Angiotensin converting enzyme (ACE) inhibitory peptide from the tuna (Thunnus thynnus) muscle: Screening, interaction mechanism and stability

In this study, the purpose was to screen novel angiotensin converting enzyme inhibitory peptides (ACEIPs) from tuna muscle taking two-steps enzymatic hydrolysis (Neutrase and Alkaline). Following isolation and purification by ultrafiltration, the Sephadex G-15 gel chromatography and reversed-phase high-performance liquid chromatography based on active-guide, the amino acid sequence was identified using Q-Orbitrap-MS/MS. Five peptides were chose synthesized based on the in silico screening methods. Among these, the two novel ACEIPs LTGCP and YPKP showed better inhibitory ability, and their corresponding IC50 values were 64.3 μM and 139.6 μM. Subsequently, the interaction mechanism of the best active peptide (LTGCP) against ACE was investigated by inhibitory pattern, molecular docking and molecular dynamic simulation. The result displayed that LTGCP was a mix-type inhibitor against ACE from the Lineweaver-Burk plots. LTGCP formed seven hydrogen bonds based on the molecular docking and the binding energy was −7.29 kcal/mol. LTGCP formed a stability complex with ACE based on the molecular dynamic simulation. Besides, LTGCP exhibited good stability in various temperature, pH and gastrointestinal digestion. Finally, the 0.125 mM ∼ 1.0 mM LTGCP exhibited no-toxic for Caco-2 cell. In summary, these findings showed that tuna was a good material to prepare ACEIPs and LTGCP may be the good potential antihypertensive drug or nutraceuticals.

Affibody-based molecular probe 99mTc-(HE)3ZHER2:V2 for non-invasive HER2 detection in ovarian and breast cancer xenografts.

This study aimed to assess the biodistribution and bioactivity of the affibody molecular probe 99mTc-(HE)3ZHER2:V2, prepared by genetic recombination, and to investigate its potential for targeted human epidermal growth factor receptor 2 (HER2) imaging in SKOV3 ovarian cancer and MDA-MB-361 breast cancer xenografts. Affibody molecules were generated through genetic recombination. The radiochemical purity of the 99mTc-labeled HER2 affibody was determined using reverse phase high performance liquid chromatography (RP-HPLC). Evaluation of HER2 affinity in SKOV3 ovarian cancer cells and MDA-MB-361 breast cancer cells (HER2-positive) was conducted by calculating equilibrium dissociation constants. Biodistribution of the 99mTc-labeled affibody molecular probe was assessed in Balb/c mice bearing SKOV3 tumors. Tumor targeting specificity was evaluated in Balb/c mice using SKOV3, MDA-MB-361, and AT-3 (HER2-negative) xenografts. Affibody (HE)3ZHER2:V2, generated through recombinant gene expression, was successfully labeled with 99mTc, achieving a radiochemical purity of (96.0 ± 1.7)% (n = 3) as determined by RP-HPLC. This molecular probe exhibited specific binding to HER2-positive SKOV3 cells, demonstrating intense radioactive uptake. Biodistribution analysis showed rapid accumulation of 99mTc-(HE)3ZHER2:V2 in HER2-positive tumors post-administration, primarily clearing through the urinary system. Single-photon emission computed tomography imaging conducted 1-3 h after intravenous injection of 99mTc-(HE)3ZHER2:V2 into HER2-positive SKOV3 and MDA-MB-361 nude mouse models confirmed targeted uptake of the molecular probe by the tumors. The molecular probe 99mTc-(HE)3ZHER2:V2 developed in this study effectively targets HER2 for imaging HER2-positive SKOV3 and MDA-MB-361 xenografts in vivo. It exhibits rapid blood clearance without evident toxic effects, suggesting its potential as a valuable marker for detecting HER2 expression in tumor cells.

Suspect screening-data independent analysis workflow for the identification of arsenolipids in marine standard reference materials.

There has been limited research into arsenolipid toxicological risks and health-related outcomes due to challenges with their separation, identification, and quantification within complex biological matrices (e.g., fish, seaweed). Analytical approaches for arsenolipid identification such as suspect screening have not been well documented and there are no certified standard reference materials, leading to issues with reproducibility and uncertainty regarding the accuracy of results. In this study, a detailed workflow for the identification of arsenolipids utilizing suspect screening coupled with data independent analysis is presented and applied to three commercially available standard reference materials (Hijiki seaweed, dogfish liver, and tuna). Hexane and dichloromethane/methanol extraction, followed by reversed-phase high-performance liquid chromatography-inductively coupled plasma mass spectrometry and liquid chromatography-electrospray ionization-quadrupole time-of-flight mass spectrometry. Using the workflow developed, mass fragmentation matching, mass error calculations, and retention time matching were performed to identify suspect arsenolipids. Arseno-fatty acids (AsFAs), arsenohydrocarbons (AsHCs), and arsenosugar phospholipids (AsSugPLs) were identified with high confidence; AsHC332, AsHC360, and AsSugPL720 in seaweed, AsHC332 in tuna, and AsFA474 and AsFA502 in the dogfish liver. AsHC332, AsHC360, and AsFA502 were identified as promising candidates for further work on synthesis, quantification using MS/MS, and toxicity testing.

Harringtonine metabolites: 5'-de-O-methylharringtonine and cephalotaxine, targeting spike protein and TMPRSS2 to double block membrane fusion of SARS-CoV-2 and its variants

Membrane fusion is the main pathway for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to invade host cells. Harringtonine (HT), derived from cephalotaxus fortunei Hook. f., has been recognized as an effective antagonist of SARS-CoV-2. It can directly block the active binding of spike (S) protein to host angiotensin converting enzyme 2 (ACE2), as well as hinder the enzymolysis of transmembrane serine proteases 2 (TMPRSS2). This study examined the potential of HT metabolites, 5'-de-O-methylharringtonine and cephalotaxine, as the membrane fusion inhibitors for SARS-CoV-2. 5'-De-O-methylharringtonine was synthesized and subsequently characterized by high resolution mass spectrometry and nuclear magnetic resonance to be structurally consistent, with a purity of 92.677% determined by reverse phase high performance liquid chromatography. Both 5'-de-O-methylharringtonine and cephalotaxine can specifically bind to SARS-CoV-2 S protein and TMPRSS2 using cell membrane chromatography. They can form hydrogen bonds with key sites that correlated highly with the enhanced binding affinity of SARS-CoV-2 and its variants to ACE2 or nafamostat to TMPRSS2. Moreover, 5'-de-O-methylharringtonine and cephalotaxine can inhibit pseudotyped virus entry and membrane fusion in a dose-dependent manner, with enhanced effectiveness upon elevated expression of TMPRSS2. Importantly, they displayed low cytotoxic effects on human normal cell lines. Our study suggested that 5'-de-O-methylharringtonine and cephalotaxine were of low toxicity and safety for humans as potential antagonists of SARS-CoV-2 and its variants, which deserve further validation in a biosafety level 3 facility.

In vitro insulinotropic actions of extracts of Gnetum africanum welw and effects on glucose homeostasis in mice with diet-induced obesity-diabetes

IntroductionGnetum africanum leaf is consumed as a vegetable in many parts of Africa. Many ethnobotanical surveys indicate its usage in the management of inflammation-related diseases (such as diabetes). However, mechanisms underlying its antidiabetic actions are poorly understood. MethodsThis study conducted phytochemical analysis and investigated mechanisms underlying anti-diabetic actions of aqueous extracts of G. africanum in cellular and high fat-fed mice models of diabetes. Reversed phase high performance liquid chromatography (HPLC) analysis, qualitative phytochemical screening and the estimation of the total flavonoid and total phenolic content were conducted. Mechanisms underlying insulinotropic actions of the extracts as well as its effects on cytotoxicity, cell viability, intracellular calcium and membrane potential were assessed. In vivo actions were evaluated in mice with diet-induced obesity-diabetes. ResultsG. africanum has total phenolic content of 1.24±0.23GE/g and total flavonoid content of 1.54±0.06QE/g. HPLC analysis revealed the presence of the presence of atropine, vicenin 3, vicenin 2, alpha-chaconine, isoswertisin and solanidine. The extract stimulated non-toxic, concentration-dependent insulin-release from BRIN-BD11 cells (1.3-fold to 3.3-fold, P < 0.05 to P < 0.001). As glucose concentration increased from 1.1 mM to 5.6 mM and 5.6 mM to 16.7 mM, these effects increased by 2.0-fold (P < 0.001) and 1.2-fold (P < 0.05) respectively. Insulinotropic effects increased in cells depolarised with KCl (30 mM, 1.3-fold, P < 0.05). These effects reduced in the presence of diazoxide (300 µM, 67 %, P < 0.001), verapamil (50 nM, 50 %, P < 0.001), and absence of extracellular calcium (55 %, P < 0.001). Membrane potential (48 %, P < 0.05) and intracellular calcium (34 %, P < 0.05) increased in cells incubated with the extract. The extract improved glucose tolerance (22.2 %, P < 0.01 at 150 mg kg-1 bw; 35.1 %, P < 0.001 at 300 mg kg-1 bw) and plasma insulin levels (1.4-fold, P < 0.05 at 150 mg kg-1 bw; 2.9-fold, P < 0.001 at 300 mg kg-1 bw) in high fat fed mice in a dose-dependent manner. ConclusionThese results suggest a role for the KATP-dependent pathway in G. africanum insulinotropic actions and encourage further development of the therapeutic potential of the extract.

Hydrogen-rich solvent method enhances the extraction of phenolics, pigments, reducing sugars, organic acids, and vitamin C from cowslip (Primula veris L.) flower

Cowslip (Primula veris L.) is an antioxidant-rich plant used for many food and medicinal purposes. In this study, the effect of incorporating hydrogen (H2) into water (HRW), ethanol (HRE), and methanol (HRM) on the extraction of flavonoids (TFC), phenolics (TPC), and antioxidants (metal chelation, FRAP, DPPH, and ABTS) as well as color (L*, a*, b*, C*, H°), chlorophyll (a, b), lycopene, and β-carotene of wild cowslip flowers was evaluated. The results were submitted to Principal Component Analysis and correlation. Phenolic compounds, organic acids, vitamin C, and sugar content of the extract were analyzed using high-performance reverse phase liquid chromatography (HPLC-DAD/RID). The highest TPC, TFC, and antioxidant activity (metal chelation, FRAP, DPPH, and ABTS) were shown for HRM samples, followed by HRE samples, while the lowest were found for pure solvents. Incorporating H2 into all solvents significantly increased TPC, TFC, metal chelation, DPPH, and ABTS scavenging activity, as well as b*, C*, H°, chlorophyll, lycopene, and β-carotene values. The levels of some phenolics (catechin, epicatechin, rutin, and quercetin), organic acids (tartaric acid and succinic acid), reducing sugars (maltose, glucose, and fructose), and ascorbic acid increased by 10–90 % when an H2-rich solvent was used instead of the pure solvent. This is the first report showing the enhanced extraction of organic acids, pigments, and ascorbic acid by hydrogen-rich solvent. The main benefits of the HRS method are its sustainability and high efficiency in extracting bioactive compounds from medicinal plants.

Comparative venom analysis between melanistic and normally coloured phenotypes of the common adder (Vipera berus).

Snake venom is an ecologically relevant functional trait directly linked with a snake's fitness and survival, facilitating predation and defence. Snake venom variation occurs at all taxonomic levels, but the study at the intraspecific level is still in its early stages. The common adder (Vipera berus) exhibits considerable variation in colour phenotypes across its distribution range. Melanistic (fully black) individuals are the subject of myths and fairytales, and in German folklore such 'hell adders' are considered more toxic than their normally coloured conspecifics despite any formal investigation. Here, we provide the first comparative analysis of venoms from melanistic and normally coloured common adders. Specifically, we compared the venom profiles by sodium dodecylsulfate polyacrylamide gel electrophoresis and reversed-phase high-performance liquid chromatography and tested the venoms' protease, phospholipase A2 and cytotoxic activities. Phospholipase A2 activity was similar in both phenotypes, whereas general protease activity was higher in the melanistic venom, which was also more cytotoxic at two concentrations (6.25 and 12.5 µg ml-1). These minor differences between the venoms of melanistic and normally coloured adders are unlikely to be of clinical relevance in the context of human envenomation. In light of our results, the claim that melanistic adders produce more toxic venom than their normally coloured conspecifics appears rooted entirely in folklore.

Influence of hydrophilicity and shielding degree on the chromatographic properties of polyelectrolyte-grafted mixed-mode stationary phases

The series of mixed-mode stationary phases with varying polyelectrolyte hydrophilicity and attachment density to poly(styrene–divinylbenzene) surfaces was synthesized. The influence of resins hydrophilicity and substrate shielding degree on the chromatographic properties of obtained phases in ion chromatography (IC), hydrophilic interaction liquid chromatography (HILIC), and reversed-phase high-performance liquid chromatography (RP-HPLC) modes was investigated. The stationary phase with more hydrophilic amine, namely N-methylethanolamine, in polyelectrolyte structure allowed simultaneous separation of highly polarizable anions and standard inorganic ones in IC mode, various polar analytes in HILIC mode, and nitro- and chloro-phenols as well as alkylbenzenes in RP-HPLC mode. Covering the substrate surface with polyethyleneimine prior to polyelectrolyte attachment turned out to be the most effective way to increase the hydrophilicity and shielding degree. The application of this approach resulted in growing the separation ability toward nucleosides and nucleobases as compared to previously obtained mixed-mode stationary phases based on the same substrate. The retention mechanism of phenols on polymer-based polyelectrolyte-grafted resins was studied for the first time.

Structure and functional studies of Avt1, a novel peptide from the sea anemone Aulactinia veratra

Sea anemones are a rich source of peptide toxins spanning a diverse range of biological activities, typically targeting proteins such as ion channels, receptors and transporters. These peptide toxins and their analogues are usually highly stable and selective for their molecular targets, rendering them of interest as molecular tools, insecticides and therapeutics. Recent transcriptomic and proteomic analyses of the sea anemone Aulactinia veratra identified a novel 28-residue peptide, named Avt1. Avt1 was produced using solid-phase peptide synthesis, followed by oxidative folding and purification of the folded peptide using reversed-phase high-performance liquid chromatography. The liquid chromatography-mass spectrometry profile of synthetic Avt1 showed a pure peak with molecular mass 6 Da less than that of the reduced form of the peptide, indicating the successful formation of three disulfide bonds. The solution structure determined by NMR revealed that Avt1 adopts an inhibitor cystine knot (ICK) fold, in which a ring is formed by two disulfide bonds with a third disulfide penetrating the ring to create the pseudo-knot. This structure provides ICK peptides with high structural, thermal and proteolytic stability. Consistent with its ICK structure, Avt1 was resistant to proteolysis by trypsin, chymotrypsin and pepsin, although it was not a trypsin inhibitor. Avt1 at 100 nM showed no activity in patch–clamp electrophysiological assays against several mammalian voltage-gated ion channels, but has structural features similar to toxins targeting insect sodium ion channels. Although sequence homologues of Avt1 are found in a number of sea anemones, this is the first representative of this family to be characterised structurally and functionally.

Characterization of Siderophore Produced by Bacillus velezensis YL2021 and Its Application in Controlling Rice Sheath Blight and Rice Blast.

Bacillus velezensis YL2021 has extensive antimicrobial activities against phytopathogens, and its genome harbors a catechol-type siderophore biosynthesis gene cluster. Here, we describe the characterization of siderophore produced by strain YL2021 and its antimicrobial activity in vitro and in vivo. A few types of siderophores were detected by chrome azurol S plates coupled with Arnow's test, purified and identified by Reversed-phase high-performance liquid chromatography (RP-HPLC). We found that strain YL2021 can produce different antimicrobial compounds under low-iron M9 medium or iron-sufficient LB medium although antimicrobial activities can be easily observed on the two media as described above in vitro. Strain YL2021 can produce at least three catechol-type siderophores in low-iron M9 medium while no siderophore was produced in LB medium. Among them, the main antimicrobial siderophore produced by strain YL2021 was bacillibactin, with m/z of 882, based on the liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, which has broad-spectrum antimicrobial activities against Gram-positive, Gram-negative bacteria, the oomycete Phytophthora capsici and phytopathogenic fungi. Moreover, the antifungal activity of siderophore including bacillibactin observed in vitro was correlated with control efficacies against rice sheath blight disease caused by Rhizoctonia solani and rice blast disease caused by Magnaporthe oryzae in vivo. Collectively, the results demonstrate that siderophore including bacillibactin produced by Bacillus velezensis YL2021 is a promising biocontrol agent for application in rice disease control.