Reversed-phase High-performance Liquid Chromatography Research Articles

Two novel angiotensin-converting enzyme (ACE) and dipeptidyl peptidase IV (DPP-IV) inhibiting peptides from tilapia (Oreochromis mossambicus) skin and their molecular docking mechanism.

In the study, papain was used to hydrolyze tilapia (Oreochromis mossambicus) skin to obtain a tilapia skin hydrolysate (TSH) with dual angiotensin-converting enzyme (ACE) and dipeptidyl peptidase IV (DPP-IV) inhibitory activities. The resulting TSH was sequentially fractionated by ultrafiltration, size exclusion separation chromatography, and reverse-phase high-performance liquid chromatography. Its inhibitory effects on ACE and DPP-IV were determined by commercial reagent kits. Two peptides purified from TSH were identified as Gly-Pro-Leu-Gly-Ala-Leu (GPLGAL) and Lys-Pro-Ala-Gly-Asn (KPAGN) by the ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Inhibitory concentration (IC50) of GPLGAL on ACE and DPP-IV were 117.20±1.69 and 187.10±2.75µM, respectively. IC50 of KPAGN on ACE and DPP-IV were 137.40±2.33 and 259.20±2.85µM, respectively. The molecular simulation demonstrated that the binding affinities of GPLGAL to ACE and DPP-IV proteins were -8.5 and -7.4kcal/mol, respectively, whereas those of KPAGN to ACE and DPP-IV proteins were -7.9 and -6.7kcal/mol, respectively. GPLGAL interacted with 21 amino acid residues of the ACE active site, whereas KPAGN engaged with 19 amino acid residues. Additionally, GPLGAL interacted with 10 amino acid residues of the DPP-IV active site, whereas KPAGN engaged with 13 amino acid residues. The two peptides predominantly occupied the active sites of ACE (His513, Tyr523, and Ala354) and DPP-IV (Tyr662 and Arg125) through hydrogen bonding. This leads to the deactivation of ACE and DPP-IV. PRACTICAL APPLICATION: Accelerate tilapia skin development and high-value utilization; provide foundation for preparing the peptides with dual ACE and DPP-IV inhibiting activity.

Chronic exposures to cholinesterase-inhibiting pesticides adversely affects the health of agricultural workers in India

Biomonitoring of pesticide exposure has become a public concern because of its potential health effects. The present study investigated the acetylcholinesterase (AChE) inhibitory levels and their associated health effects in agricultural areas in Telangana, India. This cross-sectional included 341 exposed participants and 152 control participants from agricultural areas. A structured questionnaire was completed and blood and urine samples were collected to measure pesticides, dialkyle phosphate (DAP) metabolites, and AChE activity using liquid chromatography-tandem mass spectrometry and reversed-phase high-performance liquid chromatography. twenty-eight pesticides were detected in blood samples at concentrations ranging 0.42-45.77 ng/mL. Six DAP metabolites were also measured in urine, and all DAP metabolites were significantly higher in the exposed group. AChE activity is significantly reduced in individuals exposed for >10 years, raising concerns regarding possible neurological disorders. These results emphasise the urgent need to investigate the health effects of pesticides exposure, especially in agriculture.

Comparative evaluation of liquid-liquid extraction and nanosorbent extraction for HPLC-PDA analysis of cabazitaxel from rat plasma

A precise, sensitive, accurate, and validated reverse-phase high-performance liquid chromatography (RP-HPLC) method with a bioanalytical approach was utilized to analyze Cabazitaxel (CBZ) in rat plasma. Comparative research on extraction recoveries was performed between traditional liquid-liquid extraction (LLE) and synthesized graphene oxide (GO) based magnetic solid phase extraction (GO@MSPE). The superparamagnetic hybrid nanosorbent was synthesized using the combination of iron oxide and GO and subsequently applied for extraction and bioanalytical quantification of CBZ from plasma by (HPLC-PDA) analysis. Fourier- transform infrared spectroscopy (FT-IR), particle size, scanning electron microscopy (SEM), and x-ray diffraction (XRD) analysis were employed in the characterization of synthesized GO@MSPE nanosorbent. The investigation was accomplished using a shim pack C18 column (150 mm×4.6 mm, 5 µm) with a binary gradient mobile phase consisting of formic acid: acetonitrile: water (0.1:75:25, v/v/v) at a 0.8 mL/min flow rate, and a λmax of 229 nm. The limits of detection (LOD) and quantitation (LOQ) have been determined to be 50 and 100 ng/mL for both LLE and SPE techniques. The linearity range of the approach encompassed from 100 to 5000 ng/mL and was found to be linear (coefficient of determination > 0.99) for CBZ. The proposed method showed extraction recovery of 76.8–88.4% for the synthesized GO@MSPE and 69.3–77.4% for LLE, suggesting that the proposed bioanalytical approach was robust and qualified for all validation parameters within the acceptable criteria. Furthermore, the developed hybrid GO@MSPE nanosorbent with the help of the proposed RP-HPLC method, showed a significant potential for the extraction of CBZ in bioanalysis.

A Complex-Type N-Glycan-Specific Lectin Isolated from Green Alga Halimeda borneensis Exhibits Potent Anti-Influenza Virus Activity.

Marine algal lectins specific for high-mannose N-glycans have attracted attention because they strongly inhibit the entry of enveloped viruses, including influenza viruses and SARS-CoV-2, into host cells by binding to high-mannose-type N-glycans on viral surfaces. Here, we report a novel anti-influenza virus lectin (named HBL40), specific for complex-type N-glycans, which was isolated from a marine green alga, Halimeda borneensis. The hemagglutination activity of HBL40 was inhibited with both complex-type N-glycan and O-glycan-linked glycoproteins but not with high-mannose-type N-glycan-linked glycoproteins or any of the monosaccharides examined. In the oligosaccharide-binding experiment using 26 pyridylaminated oligosaccharides, HBL40 only bound to complex-type N-glycans with bi- and triantennary-branched sugar chains. The sialylation, core fucosylation, and the increased number of branched antennae of the N-glycans lowered the binding activity with HBL40. Interestingly, the lectin potently inhibited the infection of influenza virus (A/H3N2/Udorn/72) into NCI-H292 cells at IC50 of 8.02 nM by binding to glycosylated viral hemagglutinin (KD of 1.21 × 10-6 M). HBL40 consisted of two isolectins with slightly different molecular masses to each other that could be separated by reverse-phase HPLC. Both isolectins shared the same 16 N-terminal amino acid sequences. Thus, HBL40 could be useful as an antivirus lectin specific for complex-type N-glycans.

Determining flavonoids in the skin of Citrus reticulata

n this work we studied the flavonoid composition of tangerine skins using 10 kinds of tangerines from various producers. The tangerine skins were peeled from fruit purchased from the market in Belgorod and dried at room temperature outside direct sunlight. The compositions of “green” ethanol-based extraction solutions and the “weighed portion of plant material: volume of extraction solution” ratios were chosen for extraction to ensure a high yield of flavonoids. For separation, reverse-phase HPLC on the Kromasil 100-5C18 stationary phase was used in environmentally friendly mobile phase compositions that contained ethanol as an organic modifier. In gradient elution mode all mobile phases contained 0.2 vol. % orthophosphoric acid. To assign extract components, we used samples of a number of substances and parameters of electronic absorption spectra in comparison with published data. It was found that the chromatographic profiles of the extracts of the studied samples were noticeably different. A number of the least lipophilic compounds, derivatives of cinnamic acids, were found in all extracts. After that, we discovered two flavanone glycosides, the main of which was hesperidin (hesperitin-7-rutinoside) with a content of is 26.0-39.9 mg per 1 g of dried raw material, and naringin (naringenin-7-neohesperidiside) in significantly smaller quantities, from 0 to 1.15 mg per 1 g. But of greatest interest were polymethoxylated flavones, among which the main component was nobiletin (5,6,7,8,3′,4′-hexamethoxyflavone) with a content from 0.03 to almost 11 mg/g in dried peel and tangeretin (5,6,7,8,4′-pentamethoxyflavone), found in slightly smaller quantities, 0.1-5.4 mg/g. It was established that nobiletin and tangeretin were mostly accumulated in the skins of small tangerines. On the whole, tangerine skin is a great source of highly biologically active flavonoids, so its processing is an important task for the food industry.

Compounds isolation from Syzygium cumini leaf extract against the Vibrio species in shrimp through bioassay-guided fractionation

This study was conducted to isolate and identify the bioactive compounds from the ethanolic extract of Syzygium cumini leaf against Vibrio species through a bioassay-guided fractionation. The ethanol extract was exposed to silica gel chromatography followed by reversed phase HPLC to isolate the most effective fraction against V. parahaemolyticus. Using further UHPLC-orbitrap-ion trap mass spectrometry, five compounds were isolated with broad-spectrum potency against a range of Vibrio species viz. V. parahaemolyticus, V. alginolyticus, V. harveyi, V. vulnificus and V. anguillarum. The IC50 values for the compounds ranged from 8 to 48 µg/mL against the most sensitive species V. vulnificus and 58 to >400 µg/mL against V. alginolyticus. The results of the toxicity tests demonstrated that the compounds were not harmful for shrimp. The study’s findings indicate that S. cumini leaf extract may contain bioactive molecules that are able to be substituted for antibiotics to treat vibriosis in shrimp farming.

Bioanalytical method development and validation for the quantification of raloxifene hydrochloride from mice plasma by RP-HPLC

Raloxifene hydrochloride (RLX) belongs to the Selective estrogen receptor modulator (SERM) class of drugs, exhibiting diverse affinity to estrogen receptors. The drug is known for its anti-cancer and anti-osteoporosis activity. The current study employs a Reverse Phase High-Performance Liquid Chromatography (RP-HPLC) method that is sensitive and time-efficient for quantitative evaluation of RLX from mice plasma, using tamoxifen citrate as an internal standard (IS). The protein precipitation technique was used to extract the RLX and IS. RLX was separated using a C8 column with an isocratic elution of a mobile phase consisting of acetonitrile and Milli-Q water (pH 3) at a 1 mL/min flow rate. The ultraviolet (UV) detection of the analyte and IS were carried out at 287 nm. The linearity plot was constructed in the range of 100–3200 ng/mL with a correlation coefficient of r2 ≥ 0.9996. The inter-day and intra-day accuracy and precision were found to be within acceptable limits. Stability studies revealed that the analyte remained stable. The validated RP-HPLC method was successfully applied to quantify RLX after oral administration in mice, and the pharmacokinetic (PK) parameters were established. The present RP-HPLC method could be a beneficial tool for the fast and reliable estimation of future PK interactions, PK analysis of formulations, and therapeutic drug monitoring.

Two-dimensional chiral metal-organic framework nanosheets L-hyp-Ni/Fe@SiO2 composite for HPLC separation

In this study, we have synthesised a chiral l-hyp-Ni/Fe@SiO2 composite as a chiral stationary phase (CSP) for high-performance liquid chromatography (HPLC) for the first time. This was achieved by coating two-dimensional (2D) chiral metal-organic framework nanosheets (MONs) l-hyp-Ni/Fe onto the surface of activated SiO2 microspheres using the “wrapped in net” method. The separation efficiency of the l-hyp-Ni/Fe chromatographic column was systematically evaluated in normal-phase HPLC (NP-HPLC) and reversed-phase HPLC (RP-HPLC) configurations, employing various racemates as analytes. The findings revealed that 16 chiral compounds were separated using NP-HPLC, and five were separated using RP-HPLC, encompassing alcohols, amines, ketones, esters, alkanes, ethers, amino acids and sulfoxides. Notably, the resolution (Rs) of nine chiral compounds exceeded 1.5, indicating baseline separation. Furthermore, the resolution performance of the l-hyp-Ni/Fe@SiO2-packed column was compared with that of Chiralpak AD-H. It was observed that certain enantiomers, which either could not be resolved or were inadequately separated on the Chiralpak AD-H column, attained separation on the 2D chiral MONs column. These findings suggest a complementary relationship between the two columns in racemate separation, with their combined application facilitating the resolution of a broader spectrum of chiral compounds. In addition, baseline separation was achieved for five positional isomers on the l-hyp-Ni/Fe@SiO2-packed column. The effects of the analyte mass and column temperature on the resolution were also examined. Moreover, during HPLC analysis, the l-hyp-Ni/Fe columns demonstrated commendable repeatability, stability and reproducibility in enantiomer separation. This research not only advances the utilisation of 2D chiral MONs as CSPs but also expands their applications in the separation sciences.

Updated component analysis method for naturally occurring sophorolipids from Starmerella bombicola

Sophorolipids (SLs) are promising glycolipid biosurfactants as they are easily produced and functional. SLs from microorganisms are comprised of mixtures of multiple derivatives that have different structures and properties, including well-known acidic and lactonic SL (ASLs and LSLs, respectively). In this study, we established a method for analyzing all SL derivatives in the products of Starmerella bombicola, a typical SL-producing yeast. Detailed component analyses of S. bombicola products were carried out using reversed-phase high-performance liquid chromatography and mass spectrometry. Methanol was used as the eluent as it is a good solvent for all SL derivatives. With this approach, it was possible to not only quantify the ratio of the main components of ASL, LSL, and SL glycerides but also confirm trace components such as SL mono-glyceride and bola-form SL (sophorose at both ends); notably, this is the first time these components have been isolated and identified successfully in naturally occurring SLs. In addition, our results revealed a novel SL derivative in which a fatty acid is bonded in series to the ASL, which had not been reported previously. Using the present analysis method, it was possible to easily track compositional changes in the SL components during culture. Our results showed that LSL and ASL are produced initially and that SL glycerides accumulate from the middle stage during the fermentation process.Key points• An easy and detailed component analysis method for sophorolipids (SLs) is introduced.• Multiple SL derivatives were identified different from known SLs.• A novel hydrophobic acidic SL was isolated and characterized.Graphical

Exploring the phytochemical and biological properties of Salsola foetida: a promising wild plant from Southeastern Algeria

ABSTRACT This study presents an initial exploration of the phytochemical composition and biological properties of Salsola foetida, a wild plant native to southeastern Algeria. The investigation encompassed an analysis of the biochemical content, phytochemical profile, and bioactivity of the plant’s aerial parts. The biomass of S. foetida was found to comprise 51.2 ± 0.4% ash, with elemental analysis revealing the presence of carbon, oxygen, calcium, sodium, chlorine, magnesium, potassium, and sulfur. Carbohydrates were identified as the predominant constituents, surpassing the levels of proteins and lipids. Phytochemical screening, coupled with Fourier Transform Infrared (FT-IR) analysis, confirmed the presence of various secondary metabolites, including alkaloids and phenols. Reversed-phase high-performance liquid chromatography (RP-HPLC) analysis further delineated a spectrum of phenolic compounds, with rutin emerging as the predominant compound at 1.59 µg/ml. Quantification revealed a noteworthy phenolic content of 67.1 ± 0.2 µg GE/mg ED. The extract exhibited robust radical scavenging effects, with the radical 50% inhibitory concentration (IC50) being 61.8 ± 0.2 μg/ml, along with protective effects against H2O2-induced hemolysis and the ability to reduce ferric ions. Additionally, the extract demonstrated anti-inflammatory, antibacterial, and antidiabetic activities. Notably, it exhibited considerable sunscreen properties, boasting a sun protected factor (SPF) value of 21 ± 1. Overall, S. foetida presents promising antioxidant activity and a spectrum of potential biological activities, underscoring its significance as a novel antioxidant candidate for applications in animal nutrition, medicine, and natural product research.

Novel EDTA mediated ethanol protein precipitation method and the application for polysorbate quantification in high protein concentration biopharmaceuticals

Non-ionic surfactants such as Polysorbate 20/ 80 (PS20/ PS80), are commonly used in protein drug formulations to increase protein stability by protecting against interfacial stress and surface absorption. Polysorbate is susceptible to degradation which can impact product stability, leading to the formation of sub-visible and/or visible particles in the drug product during its shelf-life, affecting patient safety and efficacy. Therefore, it is important to monitor polysorbate concentration in drug product formulations of biotherapeutic drugs. The common method for measuring polysorbate concentration in drug product formulations uses mixed mode ion exchange reversed phase HPLC (MAX) coupled to evaporative light scattering detection (ELSD). However, high protein concentration can adversely impact method performance due to high sample viscosity, gel formation, column clogging, interfering peaks and loss of accuracy. To overcome this, a new method was developed based on EDTA mediated ethanol protein precipitation (EDTA/EtOH). This method was successfully implemented for the analysis of polysorbate in antibody formulations with wide range of protein concentration (10–250 mg/mL).

Structural and Biochemical Characterization of a New Phage-Encoded Muramidase, KTN6 Gp46.

Endolysins are phage-encoded lytic enzymes that degrade bacterial peptidoglycan at the end of phage lytic cycles to release new phage particles. These enzymes are being explored as an alternative to small-molecule antibiotics. The crystal structure of KTN6 Gp46 was determined and compared with a ColabFold model. Cleavage specificity was examined using a peptidoglycan digest and reversed-phase high-performance liquid chromatography coupled to mass spectrometry (HPLC/MS). The structure of KTN6 Gp46 could be determined at 1.4 Å resolution, and key differences in loops of the putative peptidoglycan binding domain were identified in comparison with its closest known homologue, the endolysin of phage SPN1S. Reversed-phase HPLC/MS analysis of the reaction products following peptidoglycan digestion confirmed the muramidase activity of Gp46, consistent with structural predictions. These insights into the structure and function of endolysins further expand the toolbox for endolysin engineering and explore their potential in enzyme-based antibacterial design strategies.

Quantitative comparison and evaluation between aerial and underground parts of Gentiana straminea through simultaneous determination of five major compounds by RP-HPLC

Herbal Gentiana straminea Maxim. (Family Gentianaceae), “Ma Hua Jiao” in Chinese, is a commonly used Chinese medicine. Secoiridoids and flavonoids have been identified as the major active components of herbal medicines used in the treatment of hepatitis, rheumatism and many other diseases. It is the overharvesting of the roots of this plant for medicinal purposes that has led to a drastic decline in its population. In the present study, the above and below ground parts of Gentian Bitter Glycine were quantitatively compared and evaluated for the determination of the major active constituents. Five major compounds, loganic acid, swertiamarin, gentiopicroside, sweorside and isoorientin, were extracted by solvent extraction technique and analyzed by Reversed-phase High Performance Liquid Chromatography (RP-HPLC). By analysing the principal components and calculating the composite scores, the results show that the aboveground component in different areas ranked higher compared to the underground component, with the former being able to substitute to some extent for the latter's underground component. Finally, based on hierarchical cluster analysis, we identified the ideal natural growing region for aerial parts of G. straminea distributed on the Qinghai-Tibetan Plateau. The significance of this work is that we can balance the demand for herbs with environmental preservation by selectively picking the aerial parts, which can regrow next year, instead of removing the whole plant. It protects the fragile ecological environment of the Tibetan Plateau and is important for sustainable development.

Developed and Validated for the Estimation of Bupropion and Dextromethorphan in a Fixed Dose Combination of the Tablet.

The aim of this study was to develop a simple, accurate, and precise method for the estimation of bupropion and dextromethorphan in a fixed-dose combination of tablets and robust high-performance liquid chromatography for assay analysis of such a fixed combination. Chromatographic analysis was performed and separations were achieved on a Denali C18 150 × 4.6 mm, 5 micron using a mobile phase composition of ortho-phosphoric acid and acetonitrile in the ratio of 600:400 (v/v), flow rate of 1.0 mL/min, injection volume is 10 µL and run time of 6 min in isocratic elution. Ultraviolet (UV) detection was performed at a wavelength of 221 nm. The temperature was maintained at 30 °C. Well-resolved peaks were observed with a high number of theoretical plates, lower tailing factor, and reproducible relative retention time. The method was validated, and all validation parameters were found to be within the acceptance limits. A simple, accurate, and precise method has been developed for estimating bupropion and dextromethorphan in a fixed dose combination of tablets. The optimized method included the following parameters: column temperature of 30 °C, 40% acetonitrile as the mobile phase, and flow rate of 1.0 mL/min. Retention times were 2.25 min and 3.12 min for bupropion and dextromethorphan, respectively. The method was found to be linear in the range of 17.5-105 µg/mL [for R2 < 0.999) and 7.5-45 µg/mL (for R2 > 0.999] for bupropion and dextromethorphan, respectively. Both active pharmaceutical ingredients dissolved more than 90% within 5 min. The current study describes a new, simple, reliable, and economical elution reversed-phase high performance liquid chromatography method for estimating bupropion and dextromethorphan in a fixed combination tablet dosage form. The forced degradation studies were conducted using several degradation conditions such as acidic, alkali, oxidation, thermal, UV, and neutral conditions; the proposed method was effectively employed from the resolution of sample peaks. To the best of our knowledge, no such detailed and stability-indicating method has been reported for a fixed tablet dosage form.

Validation of HPLC-DAD Based Stability Indicating Assay Method for Ornidazole in Periodontal Polymeric Hydrogel: Robustness Study using Quality by Design (QbD) Approach

In the present study, a gradient reversed-phase high-performance liquid chromatography (RP-HPLC) method has been designed and validated to quantify Ornidazole (OZ) in the marketed formulation (oral gel) with the application of QbD. The proposed HPLC method utilizes an RP C18 column (4.6 x 250 mm, 5 μm). The mobile phase contained HPLC grade Solvent A (water) and Solvent B (acetonitrile) and ran at a flow rate of 1 mL/min in a gradient mode. The linearity was plotted in an OZ range of 1–12 μg/mL and the correlation coefficient was found to be 0.9998. The limit of quantification, as well as the limit of detection, has been recorded as 0.70 μg/mL and 0.23 μg/mL. Inter- and intra-day precision (RSD) percentages were reported respectively within a range of 0.262–0.589 and 0.179–0.879. At 80, 100, and 120 percent different OZ levels, the average accuracy was 99.55, 99.58, and 99.92, respectively. The result of the study indicates that the OZ gel estimate HPLC method has good sensitivity, reproducibility, accuracy, and precision. This method may be used for the estimation of OZ in a gel formulation in quality control laboratories.

Design and Escherichia coli Expression of a Natively Folded Multi-Disulfide Bonded Influenza H1N1-PR8 Receptor-Binding Domain (RBD).

Refolding multi-disulfide bonded proteins expressed in E. coli into their native structure is challenging. Nevertheless, because of its cost-effectiveness, handiness, and versatility, the E. coli expression of viral envelope proteins, such as the RBD (Receptor-Binding Domain) of the influenza Hemagglutinin protein, could significantly advance research on viral infections. Here, we show that H1N1-PR8-RBD (27 kDa, containing four cysteines forming two disulfide bonds) expressed in E. coli and was purified with nickel affinity chromatography, and reversed-phase HPLC was successfully refolded into its native structure, as assessed with several biophysical and biochemical techniques. Analytical ultracentrifugation indicated that H1N1-PR8-RBD was monomeric with a hydrodynamic radius of 2.5 nm. Thermal denaturation, monitored with DSC and CD at a wavelength of 222 nm, was cooperative with a midpoint temperature around 55 °C, strongly indicating a natively folded protein. In addition, the 15N-HSQC NMR spectrum exhibited several 1H-15N resonances indicative of a beta-sheeted protein. Our results indicate that a significant amount (40 mg/L) of pure and native H1N1-PR8-RBD can be produced using an E. coli expression system with our refolding procedure, offering potential insights into the molecular characterization of influenza virus infection.

The Quality by design Approach for Analytical Method Development and Validation of the RP-HPLC Method for Estimation of Quercetin in Pure, Marketed, and Cream Formulation

ABSTRACT: A simple, precise reverse phase high performance liquid chromatographic (RP-HPLC) method has been developed and validated for the flavonoid quercetin, isolated from Tridax procumbens L. The method was validated by using Phenomenex C18 (250 x 4.6mm i.d) Column. A simple, cost effective mobile phase consisting of (ACN and 10 m/moL Phosphate buffer as mobile phase in proportion of 50:50 v/v ) pH 3, 1.0 ml/min Flow rate at 370nm by using UV Visible detector. The retention time of Quercetin was found to be 3.392 minutes. The Quercetin linearity range was found to be 05 to 25 μg/mL. The accuracy and precision of commercially available preparations and in-house cream formulations were investigated using a one-way ANNOVA test. The Percentage recovery of both formulations was found to be 99.83%, 99.88%, 99.82% and 98.92%, 98.18%, 98.86%. Robustness of analytical method was studied by using 2/3 full factorial design by using Design expert software. The Percentage assay and % RSD of marketed capsule and in house cream was found to be 98.38 %, 97.40% and 0.05273, 0.02053 respectively. The Limit of Quantitation and Limit of detection were found to be 0.9053μg/mL and 2.5435μg/mL respectively. The development method of quercetin is simple, accurate, precise, sensitive, and robust

Development of a chromatographic method for optimizing the thiol-maleimide coupling of polyoxometalate-polymer hybrids

The covalent attachment of polyoxometalates (POMs) to polymers has been developed as a strategic approach for the advancement of POM-based hybrid materials with versatile applications. In this study, we utilized thiol-maleimide Michael addition to investigate the kinetics and efficacy of the “one-to-one” conjugation between Keggin type POM and polystyrene. We explored the effects of solvent polarity, catalyst, molecular weight of PS and synthetic strategies on the reaction kinetics and efficiency, by means of reverse-phase high-performance liquid chromatography (RP-HPLC). A series of comparative analysis affirmed the superior efficiency of the one-pot method, particularly when facilitated by the addition of a high-polarity solvent and an excess of maleimide. These findings offer valuable insights into the intricate interplay between reaction conditions, kinetics, and selectivity in thiol-maleimide reactions of POMs and polymers. They hold profound implications for advancing the study of POM-based multifunctional materials and the synthesis of complex hybrid molecules.

Separation of inorganic anions on reversed-phase C18 columns with a phosphomolybdate mobile phase

Reversed-phase high performance liquid chromatography (RP-HPLC) is the most widely used chromatographic method. In addition to hydrophobic interactions, additional interactions such as electrostatic interactions may participate in the retention behaviour of an analyte. This makes it possible to use RP-HPLC for many types of analyte. We describe a simple method for separating inorganic anions on a C18 column, in which retention of inorganic anions is almost entirely due to electrostatic interactions. This leads to rapid separations as well as higher theoretical plate numbers. We used 2 mM phosphoric acid containing a low concentration of disodium molybdate as the mobile phase, which allows UV detection of non-UV-absorbing anions. With this method, we determined eight inorganic anions including several non-UV-absorbing anions photometrically at 220 nm. The detection limits of the examined eight inorganic anions calculated at a signal-to-noise ratio of 3 were between 0.3 and 10 μM. The detector response was linear over three orders of magnitude of inorganic anion concentration. The proposed RP-HPLC/UV method was successfully applied to determine inorganic anions in some water samples.

Effect of high-hydrostatic pressure on the digestibility of egg yolk and granule.

The impact of high hydrostatic pressure (HHP) on protein digestibility of egg yolk and egg yolk granule was evaluated by static in vitro digestion using the standardized INFOGEST 2.0 method. The degree of hydrolysis (DH) and the phospholipid content were determined during digestion, and the protein and peptide profiles were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reverse phase-high pressure liquid chromatography (RP-HPLC). The results showed that HHP induced protein aggregation in egg yolk and granule, mainly by disulfide bridges, which were not disrupted in the oral phase. Proteolysis during the gastric phase improved egg yolk and granule protein solubility, regardless of whether HHP was applied. However, the extent of the samples' digestibility was not affected, with DH values ranging from 15% to 20%. During the intestinal phase, the DH of egg yolk protein (∼40%) was higher than that of the granule (∼25%), probably due to the denser structure of the granule reducing the accessibility of intestinal enzymes. The DH, peptide, and protein profiles of control and HHP-treated egg yolk showed similar protein digestion behaviors for both gastric and intestinal phases. Among the different proteins, only the digestibility of β-phosvitin in HHP-treated granule was enhanced. Consequently, applying HHP to granules represents an interesting process that improves the digestibility of phosvitin with the potential to generate bioactive phosvitin-derived phosphopeptides. PRACTICAL APPLICATION: High hydrostatic pressure, mainly used as a preservation process, did not impair the nutritional quality of the egg yolk and granule proteins but improved the susceptibility of phosvitin (protein contained in egg yolk) proteolysis to produce bioactive phosphopeptides. Consequently, applying HHP to granules represents an interesting process that improves the digestibility of phosvitin.