Size-exclusion High-performance Liquid Chromatography Research Articles

Structural Characterization of RC28-E, a Recombinant Fusion Protein With Dual Targets on VEGF and FGF2

Vascular endothelial growth factor (VEGF) and fibroblast growthfactor (FGF) play important roles in angiogenesis-related diseases. RC28-E is a soluble fusion protein composed of the human VEGF receptor 1 (VEGFR1) extracellular domain 2 (ECD 2), VEGFR2 ECD 3, FGFR1 ECDs 2 and 3, and the Fc regions of human immunoglobulin G1. By targeting both VEGF and FGF2, RC28-E may represent a useful antiangiogenetic agent, but structural and functional characterizations of this fusion protein are needed. Liquid chromatography–tandem mass spectrometry, size exclusion high-performance liquid chromatography, capillary electrophoresis-sodium dodecyl sulfate, imaged capillary isoelectric focusing, and bio-layer interferometry were used to characterize the properties of RC28-E. The purity of RC28-E was confirmed to be 98% or greater. The glycosylation modification of RC28-E was found to be very complicated, with 11 potential N-linked glycosylation points and 23 types of N-glycans, causing high heterogeneity of the protein. The primary modifications of the amino acid sequence of RC28-E protein included C-terminal K truncation, N-deamidation, and M-oxidation modification. Notably, RC28-E demonstrated a higher affinity for both VEGF and FGF2 than VEGF trap or FGF trap for their respective targets.

Characterization of Grinding-Induced Subvisible Particles and Free Radicals in a Freeze-Dried Monoclonal Antibody Formulation.

The primary objectives of this study were to investigate the degradation mechanisms of freeze-dried monoclonal antibody (mAb) formulations under mechanical grinding, assess the sensitivity and suitability of various particle analysis techniques, analyze the structure of the collected subvisible particles (SbVPs), and analyze the antioxidant mechanism of methionine (Met) under degradation process to gain a thorough understanding of the phenomenon. The freeze-dried mAb-X formulations underwent grinding, and the resultant SbVPs were characterized through visual inspection, flow imaging microscopy, dynamic light scattering, ultraviolet-visible spectroscopy, and size-exclusion high-performance liquid chromatography. We further evaluated the effect of different temperatures and the free radical scavenger Met on SbVP formation. The produced free radicals were detected using electron paramagnetic resonance, and Met S-oxide formation was detected using liquid chromatography-mass spectrometry. In addition, we analyzed the obtained SbVPs using capillary electrophoresis sodium dodecyl sulfate and Fourier transform infrared spectroscopy. Grinding leads to SbVP formation under high temperature and free radical formation. Free radicals produced during grinding require the participation of a macromolecule. Met could then bind to the produced free radicals, thus partially protecting mAb-X from degradation while itself undergoing oxidation to form Met(O). Sensitivity differences between different particle analysis techniques were evaluated, and the obtained SbVPs showed significant changes in secondary structure and the formation of covalent aggregates and fragments. Met plays the role of an antioxidant in protecting macromolecules by quenching the free radicals produced during grinding. To thoroughly characterize SbVPs, multiple and orthogonal particle analysis techniques should be used, and if necessary, SbVPs should be processed by enrichment to accurately analyze primary and high order structures.

Preventing the browning of fresh wet noodle sheets by aqueous ozone mixing: Browning and physicochemical properties

The formation and prevention mechanism of the dark spots in fresh wet noodle sheets (FWNS) has been discussed in our previous study, while the factors which cause FWNS browning are very complicated. In this work, the browning of FWNS was prevented through aqueous ozone mixing. Meanwhile, low field nuclear magnetic resonance (LF-NMR), size-exclusion high-performance liquid chromatography (SE-HPLC), dynamic rheometer, and scanning electron microscope (SEM) were used to measure the physicochemical properties and microstructure of the FWNS. The relationship between browning and the physicochemical properties was discussed. The results showed that aqueous ozone mixing improved the color of FWNS, in which physical factors and non-covalent bonds played a key role. The results of the LF-NMR tests showed that aqueous ozone mixing reduced the moisture degree of freedom. The results of the SE-HPLC tests showed that the moderate oxidizing capability of aqueous ozone promoted the cross-linking of disulfide bonds in FWNS. The results of the dynamic rheology tests and SEM observation showed that aqueous ozone mixing strengthened the gluten network structure of FWNS. Thus, aqueous ozone mixing made FWNS more compact and uniform, and indirectly reduced the browning rate.

Physicochemical stability of PF-05280014 (trastuzumab-qyyp; TrazimeraTM), a trastuzumab biosimilar, under extended in-use conditions.

The stability and functional activity of the trastuzumab biosimilar PF-05280014 (trastuzumab-qyyp; TrazimeraTM), was assessed under extended in-use conditions. PF-05280014 was diluted in 0.9% sodium chloride to final concentrations of 0.2 mg/mL and 4 mg/mL in 3 different types of infusion bags (polyolefin, ethylene vinyl acetate, and polyvinyl chloride). Infusion bags containing diluted PF-05280014 were stored at 25 ± 5° C for 24 h, before storage at 5 ± 3° C for 0, 1, 2, 4, or 6 weeks. Following extended storage, samples of PF-05280014 were removed from the infusion bags and stored at 25 ± 5° C for 24 h before biophysical and functional characterization. In addition to the visual characteristics of each sample at the various time points, the stability of PF-05280014 was assessed using a variety of biophysical techniques, including size-exclusion high-performance liquid chromatography, non-reducing sodium dodecyl sulfate capillary electrophoresis, cation-exchange chromatography, peptide mapping, far-UV circular dichroism spectroscopy, and differential scanning calorimetry. The functional activity of PF-05280014 was evaluated using a cell-based growth inhibition assay. For all PF-05280014 concentrations, time points and infusion bags tested, there were no significant differences in visual characteristics or in protein concentration. The were no significant changes in the relative abundance of molecular weight or charge variants throughout the 6-week study period. Similarly, there were no significant changes in primary structure or in secondary structure content during the study. The relative potency of PF-05280014 was also maintained throughout the 6-week period. The stability and functional activity of PF-05280014 was maintained following dilution in 0.9% sodium chloride and storage for up to 6 weeks at 2-8° C.

Innovative Green Way to Design Biobased Electrospun Fibers from Wheat Gluten and These Fibers' Potential as Absorbents of Biofluids.

In this study, a new method was developed to successfully design sustainable microfibers from wheat gluten proteins using a nonreducing solvent and electrospinning. We explored the morphology by X-ray tomography, scanning electron microscopy (SEM), and confocal laser scanning microscopy (CLSM), protein chemistry and cross-linking by size exclusion-high-performance liquid chromatography (SE-HPLC), and secondary structure by Fourier transform infrared spectroscopy (FT-IR) of fibers containing 15 and 20% of gluten. The impact of heat (130 °C) post-treatment on the polymerization properties of fibers and their absorption performance in different biofluids were also evaluated. The fibers with 20% gluten showed a uniform architecture supported by a relatively stronger fibrous network as compared to irregular and brittle fibers from 15% gluten. Heat treatment of fibers increased the protein cross-linking in all electrospun fibers as compared to the non-heat-treated fibers, as evidenced by SE-HPLC. An increase in the amount of α-helices and random coils was observed in the proteins of all of the heat-treated fibers compared to the nontreated fibers by FT-IR. This suggested that the heat treatment contributed positively to the gluten protein's chemical rearrangements, e.g., aggregation, new hydrogen and isopeptide bonding, and conversion of some of the sulfhydryl groups into disulfide cross-links, contributing positively to the functional performance. The heat-treated electrospun fibers with 20% gluten showed a very attractive blood absorption capacity (323%) and reasonable stability in phosphate-buffered saline (PBS) buffer compared to 15% gluten fibers and non-heat-treated fibers. Cotton-like fiber architecture, high blood absorption capacity, and reasonable stability in PBS buffer are properties desired for absorbents of biofluids and should be further explored in healthcare and medical applications.

Evaluation of the Storage Stability and Quality Properties of Fresh Noodles Mixed with Plasma-Activated Water.

Enhancing the quality retention of fresh noodles remains challenging. In this study, we investigated the effect of dough mixing with plasma-activated water (PAW) of different activation times on the storage stability and quality characteristics of fresh noodles. It was found that the total plate count in the fresh noodles prepared by PAW (PAWN) showed no obvious inhibition during storage at 25 °C, but could be significantly reduced at 4 °C as compared with the control. The decrease in L* value and pH of the PAWN was significantly retarded during storage, indicating an enhanced storage stability. The stability time of dough mixed with PAW could be significantly improved. PAW treatment decreased the viscosity properties and setback value of starch, while enhancing the interaction of water and non-water components in fresh noodles. In addition, dynamic polymerization and depolymerization of proteins were detected in Size-Exclusion High-Performance Liquid Chromatography (SE-HPLC) profiles of PAWN. The hardness and adhesiveness of the cooked noodles decreased, while the springiness significantly increased. These results implied the potential of PAW in improving the storage stability and quality of fresh noodles.

On the aggregation of bovine serum albumin

In an attempt to elucidate the aggregation behaviour of bovine serum albumin and its modulation by salt ions, size-exclusion high-performance liquid chromatography was used and complemented by dynamic light scattering. The influence of the protein concentration, and type and concentration of inorganic salt on the aggregation of albumin in solution have been analyzed. Based on the observations herein reported an aggregation mechanism is proposed, according to which (i) some functional groups of albumin dissociate in solution forming macromolecular ions; (ii) the macromolecular ions bind with each other into large aggregates; and (iii) the salt ions establish chemical equilibria with the charges of opposing character present in the macromolecular ions’ backbone, promoting or preventing the aggregation. The present work shows that the aggregation behaviour of bovine serum albumin in solution and its modulation by salt ions can be explained by chemical concepts, with chemical equilibrium playing an essential role.

Validation of Pretreatment Methods for the In-Process Quantification of Foot-and-Mouth Disease Vaccine Antigens.

Foot-and-mouth disease (FMD), caused by the FMD virus (FMDV), is controlled by vaccine policy in many countries. For vaccine potency, the content of intact virus particles (146S antigens) is critical, and the sucrose density gradient (SDG) fractionation is the gold standard for the quantification of 146S antigens. However, this method has several drawbacks. Although size-exclusion high-performance liquid chromatography (SE-HPLC) was introduced to replace the classic method, its application is generally confined to purified samples owing to the interfering signals. Therefore, we aimed to develop optimal pretreatment methods for SE-HPLC quantification in less purified samples. Crude virus infection supernatant (CVIS) and semi-purified samples with PEG precipitation (PEG-P) were used. Chloroform pretreatment was essential to remove a high level of non-specific signals in CVIS, whereas it caused loss of 146S antigens without the distinctive removal of non-specific signals in PEG-P. Benzonase pretreatment was required to improve the resolution of the target peak in the chromatogram for both CVIS and PEG-P. Through spiking tests with pure 146S antigens, it was verified that the combined pretreatment with chloroform and benzonase was optimal for the CVIS, while the sole pretreatment of benzonase was beneficial for PEG-P.

Insight into the mechanism of d-allose in reducing the allergenicity and digestibility of ultrasound-pretreated α-lactalbumin by high-resolution mass spectrometry

The mechanism of d-allose in reducing the allergenicity and digestibility of ultrasound-pretreated α-lactalbumin (α-LA) was studied. The intensity reduction and peak red shift occurred in fluorescence spectra of glycated samples. Enzyme-linked immunosorbent assay and basophil degranulation analysis showed that d-allose significantly reduced the allergenicity of α-LA, and ultrasound-pretreated α-LA showed the lowest allergenicity after glycation. Compared with α-LA, the degree of hydrolysis decreased in glycated samples. Size-exclusion high-performance liquid chromatography showed that the glycated α-LA was resistant to digestive enzymes. The glycated sites and average degree of substitution per peptide molecule were determined using LC Orbitrap MS/MS. These results suggested that the masking of linear allergenic epitopes by glycation could reduce the allergenicity. Therefore, the combination of ultrasound pretreatment and glycation is a potential method to reduce protein allergenicity in food processing and provides a useful approach for the application of rare sugars in food processing.

Citric acid chelated Fe(II) catalyzed peroxidation for simultaneously improving sludge dewaterability and antibiotic resistance genes (ARGs) removal

Sludge contains large amounts of antibiotic resistance genes (ARGs), that reduce the efficacy of antibiotic therapies when transmitted to humans. However, conventional sludge treatment often fails to remove ARGs. Herein, critic acid chelated Fe(II) catalyzed peroxidation (CA-Fenton) was conducted to simultaneously improve sludge dewaterability and ARGs removal. As results, the CA-Fenton process exhibited significantly improved sludge dewatering performance and ARGs removal, in which the capillary suction time (CST), specific resistance to filtration (SRF) and cake moisture of sludge were reduced from 8.8 s·L/g, 5.8 × 1013 m/kg and 93.2% to 2.3 s·L/g, 5.8 × 1012 m/kg and 79.1%, respectively, and the removal efficiencies of 7 types-ARGs in sludge achieved over 90%. In addition, synergistic sludge dewatering and ARGs degradation mechanisms were elucidated with a suit of macro and spectroscopic evidence (high performance liquid chromatography-size exclusion chromatography (HPLC-SEC) and two-dimensional fourier-transform infrared correlation spectroscopy (2D-FTIR)). During CA-Fenton treatment, CA acidification enhanced permeability of the cell barrier in EPS, releasing intracellular ARGs into sludge bulk solution, which accelerated the ARGs degradation via CA chelated Fe(II) catalyzed H2O2 process. Meanwhile, destruction of secondary structure represented β-turn in proteins enhanced the hydrophobicity of EPS, which contributed the improvement of sludge dewaterability. The study not only proposed an efficient method to improve traditional Fenton oxidation for sludge treatment, but also provided a mechanistic basis for better understanding of the CA-Fenton process, and enable the harmless disposal for sludge.

Investigation of Protein and Epitope Characteristics of Oats and Its Implications for Celiac Disease.

The use of pure oats (oats cultivated with special care to avoid gluten contamination from wheat, rye, and barley) in the gluten-free diet (GFD) represents important nutritional benefits for the celiac consumer. However, emerging evidence suggests that some oat cultivars may contain wheat gliadin analog polypeptides. Consequently, it is necessary to screen oats in terms of protein and epitope composition to be able to select safe varieties for gluten-free applications. The overall aim of our study is to investigate the variability of oat protein composition directly related to health-related and techno-functional properties. Elements of an oat sample population representing 162 cultivated varieties from 20 countries and the protein composition of resulting samples have been characterized. Size distribution of the total protein extracts has been analyzed by size exclusion-high performance liquid chromatography (SE-HPLC) while the 70% ethanol-extracted proteins were analyzed by RP-HPLC. Protein extracts separated into three main groups of fractions on the SE-HPLC column: polymeric proteins, avenins (both containing three subgroups based on their size), and soluble proteins, representing respectively 68.79–86.60, 8.86–27.72, and 2.89–11.85% of the total protein content. The ratio of polymeric to monomeric proteins varied between 1.37 and 3.73. Seventy-six reversed phase-HPLC-separated peaks have been differentiated from the ethanol extractable proteins of the entire population. Their distribution among the cultivars varied significantly, 6–23 peaks per cultivar. The number of appearances of peaks also showed large variation: one peak has been found in 107 samples, while 15 peaks have been identified, which appeared in less than five cultivars. An estimation method for ranking the avenin-epitope content of the samples has been developed by using MS spectrometric data of collected RP-HPLC peaks and bioinformatics methods. Using ELISA methodology with the R5 antibody, a high number of the investigated samples were found to be contaminated with wheat, barley, or rye.

Egg white protein addition induces protein aggregation and fibrous structure formation of textured wheat gluten

The effect of egg white protein addition on the fibrous structure and protein aggregation of textured wheat gluten (TWG) extrudates was investigated. The hardness, springiness, chewiness, and degree of texturization of TWG significantly increased with the addition of egg white protein. Analysis of morphological characteristics showed a positive effect of egg white protein on the formation of the fibrous structure of TWG. The results of size-exclusion high performance liquid chromatography (SE-HPLC) indicated that the egg white protein improved the degree of wheat gluten aggregation, and the analysis of the protein intermolecular forces proved that disulfide bonds were the main contributor to the cross-linking of protein. In addition, an increase in the β-sheets also indicated an increase in protein aggregation induced by egg white protein. The addition of egg white protein promoted protein interactions and improved the fibrous structure of TWG.

Physical-chemical properties and in vitro digestibility of phosphorylated and glycosylated soy protein isolate

Increased demand for highly functional soy protein isolate (SPI) in the food industry draws extra attention to SPI modification. The structural properties and in vitro digestibility of phosphorylated and glycosylated SPI were investigated in this study. The phosphorylation was carried out with sodium tripolyphosphate addition at 45 °C for 4 h. Glucose and dextran were used for glycosylation at 100 °C for 1 h. Apparent molecular weight distribution was determined by size exclusion-high performance liquid chromatography. Thiol content and surface hydrophobicity were also determined. The apparent molecular weight and thiol content of phosphorylated SPI increased with the increase of phosphorylation degree. Meanwhile, phosphorylation negatively affected gastrointestinal digestibility, especially gastric digestibility. The fraction of glycosylated SPI was prone to be low molecular weight, suggesting glycosylation inhibited SPI aggregation, which resulted from the attenuated surface hydrophobicity. The digestibility of SPI was increased by glycosylation. Glucose-based glycosylation contributed to lower intestinal digestion compared with dextran-based glycosylated SPI. This work highlighted that the phosphorylation and glycosylation to SPI primarily affected the in vitro pepsin or pancreatin digestion, inspiring to produce modified SPI with balanced functional and nutritional properties for the food industry.

Research on preparation and in vitro evaluation of the dendrimer-peptide nuclear acid conjugate for amplification pretargeting.

Amplification pretargeting has the potential to increase the tracer's accumulation in the tumor. This study aimed to develop a three-step amplification pretargeting strategy in nuclear medicine with a polymer conjugated with multiple copies of peptide nuclear acid (PNA). In this study, the tracer 18 F-labeled complementary PNA (18 F-cPNA) was prepared by click-chemistry with high radiochemical purity (>99%) and great stability in vitro. The PAMMA dendrimer generation 4 (G4) was conjugated with multiple copies of PNAs. The average number of PNA groups in the G4-PNA conjugate was determined by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and the accessibility to the 18 F-cPNA was identified by size-exclusion high-performance liquid chromatography (SE-HPLC). There were approximately 11.7 of 64 carboxyl groups modified with PNAs, of which more than 99% were accessible to 18 F-cPNA. 18 F-cPNA was added to a mixture of CC49-cPNA and G4-PNA, and the complex exhibited a single peak on high-performance liquid chromatography (HPLC) as evidence of complete hybridization between 18 F-cPNA and CC49-cPNA/G4-PNA. The LS174T tumor cells were incubated with CC49-cPNA followed by G4-PNA as an amplification platform before 18 F-cPNA was added to hybridize with CC49-cPNA/G4-PNA. Compared with conventional pretargeting without G4-PNA, the radioactivity signal was amplified about four times, which demonstrated that the dendrimer-PNA conjugate plays a crucial role in signal amplification.

Determination of free carrier protein in glycoconjugate vaccines by size exclusion chromatography–high performance liquid chromatography with fluorescence detector

AbstractPolysaccharide‐protein conjugates elicit a protective immune response in children below 2 years. The conjugated antigens using different carrier proteins generate T‐cell dependent antibody response in children and increased antibody titers in adults than unconjugated polysaccharide vaccines. Different marketed conjugate vaccines, contain carrier proteins such as cross‐reactive material, Tetanus Toxoid, Diphtheria toxoid and Protein D (derived from non‐typeable Haemophilus influenzae). Quantification of unconjugated polysaccharide (free poly) and protein (free protein) in the purified conjugates (drug substance) and formulated drug product are important quality control parameters. In addition, the bulk conjugates are monitored for free protein as stability indicating parameter. Since the purified conjugates contain low amounts of free protein, highly sensitive methods are needed. We developed a method using size‐exclusion chromatography with high‐performance liquid chromatography with fluorescence detection for free protein quantification and validated. The method can be used for quantifying the free carrier protein in Pneumococcal, Typhoid and Hib polysaccharides conjugated with different carrier proteins such as cross‐reactive material, Tetanus Toxoid and pneumococcal surface adhesin A. The method we report is sensitive, accurate, and precise with a linear range of 0.625 to 40 μg/mL and the limit of quantification is 0.12, 1.74, and 13.2 μg/mL for CRM197, PsaA, and Tetanus Toxoid, respectively. Spike recoveries were between 80 and 120% for all the conjugates using different carrier proteins. Reported method is simple, direct and can be used for quantitative determination of any free carrier protein in polysaccharide conjugates with greater application in polysaccharide‐protein conjugate manufacturing and release in the vaccine industry.

Sustainable Wheat Production and Food Security of Domestic Wheat in Tajikistan: Implications of Seed Health and Protein Quality.

Staple crop yield, quality and sustainable production are critical for domestic food security in developing countries. In Tajikistan, both seed-borne diseases and protein quality impair the yield and the quality of the major staple crop, wheat. Here, we used a detailed two-year survey of fields on 21 wheat-producing farms in Tajikistan, combined with lab analyses on seed health and protein quality, to investigate the presence of seed-borne diseases and bread-making quality in Tajik wheat. Seed samples were collected for the analysis of: (i) the presence of common bunt (Tilletia spp.) using the centrifuge wash test, (ii) the major pathogenic fungi on/in the seed using the agar plate test and (iii) the protein amount and size distribution using size-exclusion high-performance liquid chromatography (SE-HPLC). Field occurrence of common bunt and loose smut was generally low (3 farms in year one (14%) showed common bunt occurrence), but the presence of fungi was observed microscopically on most seed samples (on seeds from 19 out of 21 farms = 91%). Tilletia laevis was the dominant agent in common bunt (present in 19 farms compared to T. tritici present in 6 farms). Altogether, 18 different fungi were identified from seed samples by microscopy. Protein composition, measured with high-performance liquid chromatography as protein amount and size distribution (known to correlate with bread-making quality), differed significantly between samples from different farms and years, although the farm type and land elevation of the farm were not the determinants of the protein composition. The presence of certain fungi on the seed correlated significantly with the protein quality and could then have an impact on the bread-making quality of the Tajik wheat. The presence of seed-borne diseases, a low protein content and weak gluten were the characteristics of the majority of the grain samples, mostly irrespective of farm type and farmer’s knowledge. For sustainable development of the Tajik farming systems, and to strengthen the food security of the country, the knowledge of Tajik farmers needs to be increased independently of farm type; in general, plant breeding is required and certified seeds need to be made available throughout the country.

Innovatively processed quinoa (Chenopodium quinoa Willd.) food: chemistry, structure and end-use characteristics.

Quinoa (Chenopodium quinoa Willd.) flour and processed traditional Peruvian quinoa breakfast foods were studied to evaluate the effect of extrusion and post-processing on protein properties, morphology and nutritional characteristics (amino acids and dietary fibers). The extrusion increased quinoa protein crosslinking and aggregation observed by size exclusion high-performance liquid chromatography and the amount of soluble fibers, as well as decreasing the amounts of insoluble fibers in the processed foods. The post-processing drying resulted in additional crosslinking of large protein fractions in the quinoa products. The microstructure of the extruded quinoa breakfast flakes and heat-post-processed samples studied by scanning electron microscopy and X-ray tomography differed greatly; post-drying induced formation of aerated protein microstructures in the heat-treated samples. Nanostructures revealed by small-angle and wide-angle X-ray scattering indicated that extrusion imparted morphological changes in the quinoa protein and starch (dominance of V-type). Overall, extrusion processing only reduced the content of most of the essential amino acids to a minor extent; the content of valine and methionine was reduced to a slightly greater extent, but the final products met the requirements of the Food and Drug Organization. This study presents innovative examples on how extrusion processing and post-processing heat treatment can be used to produce attractive future food alternatives, such as breakfast cereal flakes and porridge powder, from quinoa grains. Extrusion of quinoa flour into Peruvian foods was shown to be mostly impacted by the processing temperature and processing conditions used. Protein crosslinking increased due to extrusion and post-processing heating. Starch crystallinity decreased most when the product was dried after processing. © 2021 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Rapid size-exclusion high performance liquid chromatography method for the quality control of amyloid-β oligomers

Amyloid-β (Aβ) dysmetabolism is thought to be the main trigger for neurodegenerative events in Alzheimer's disease (AD). In particular, soluble Aβ oligomers (AβOs) are proposed as key mediators of synaptic and cognitive dysfunction in AD. Over the past few decades, AβOs prepared from synthetic Aβ have been widely applied in vitro and in vivo, the so-called chemical models of AD, uncovering their multiple neurotoxic mechanisms. However, the lack of a reliable quality control (QC) for synthetic AβOs may reflect poor experimental reproducibility. In keeping with this, we optimized and validated a rapid and reproducible SECHPLC method using fluorescence detection for the QC of synthetic AβOs. Our analytical method offers an unprecedent alternative to improve the reproducibility of AD chemical models.

(-)-Epicatechin-An Important Contributor to the Antioxidant Activity of Japanese Knotweed Rhizome Bark Extract as Determined by Antioxidant Activity-Guided Fractionation.

The antioxidant activities of Japanese knotweed rhizome bark extracts, prepared with eight different solvents or solvent mixtures (water, methanol, 80% methanol(aq), acetone, 70% acetone(aq), ethanol, 70% ethanol(aq), and 90% ethyl acetate(aq)), were determined using a 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical-scavenging assay. Low half maximal inhibitory concentration (IC50) values (2.632–3.720 µg mL−1) for all the extracts were in the range of the IC50 value of the known antioxidant ascorbic acid at t0 (3.115 µg mL−1). Due to the highest extraction yield (~44%), 70% ethanol(aq) was selected for the preparation of the extract for further investigations. The IC50 value calculated for its antioxidant activity remained stable for at least 14 days, while the IC50 of ascorbic acid increased over time. The stability study showed that the container material was of great importance for the light-protected storage of the ascorbic acid(aq) solution in a refrigerator. Size exclusion–high-performance liquid chromatography (SEC-HPLC)–UV and reversed phase (RP)-HPLC-UV coupled with multistage mass spectrometry (MSn) were developed for fractionation of the 70% ethanol(aq) extract and for further compound identification, respectively. In the most potent antioxidant SEC fraction, determined using an on-line post-column SEC-HPLC-DPPH assay, epicatechin, resveratrol malonyl hexoside, and its in-source fragments (resveratrol and resveratrol acetyl hexoside) were tentatively identified by RP-HPLC-MSn. Moreover, epicatechin was additionally confirmed by two orthogonal methods, SEC-HPLC-UV and high-performance thin-layer chromatography (HPTLC) coupled with densitometry. Finally, the latter technique enabled the identification of (−)-epicatechin. (−)-Epicatechin demonstrated potent and stable time-dependent antioxidant activity (IC50 value ~1.5 µg mL−1) for at least 14 days.

Isolation of Extracellular Vesicles (EVs) Using Size-Exclusion High-Performance Liquid Chromatography (SE-HPLC).

Extracellular vesicles (EVs), are membrane-bound nanoparticles of biological origin. These signature molecules of health and disease have raised remarkable attention of the biomedical arena due to its potential diagnostic and therapeutic applicability. Among the many different techniques available for EV isolation, size-exclusion chromatography (SEC) is widely accepted.In this chapter, we present a protocol of size-exclusion high-performance liquid chromatography (SE-HPLC) as a method of EV isolation. This method can be adapted as a low cost but a reliable and scalable method of EV isolation in those laboratories having access to the HPLC systems.