Size Exclusion Chromatography Method Research Articles

Structural basis for lignin recalcitrance during sulfite pulping for production of dissolving pulp from pine heartwood

Dissolving pulps are technically produced by prehydrolysis kraft, one-stage or two-stage acid sulfite pulping. Like other pulping methods, the delignification process is incomplete and bleaching is required for complete lignin removal. Here, we explored the molecular aspects of lignin recalcitrance during the pulping, in order to gain insights that could inform future pulping efforts. For this purpose, we adopted a protocol for the controlled fractionation of pulp into soluble fractions that could be analyzed by spectroscopic methods including size exclusion chromatography (SEC) and 2D NMR methods. In addition, lignosulfonates (i.e. technical lignin) was analysed as a reference to gain insights on the structural basis for dissolution. Overall, the results identify a sequence of reactions responsible for the dissolution of lignin. In the first stage, the sulfonation of lignin begins and occurs at the α-carbon of β-O-4 and β-5 sub-structures. In the second stage the cleavage of lignin carbohydrate bonds (LCC) of benzyl ether, gamma ester and phenylglycosides types, all of which were detected in the residual lignin of the earlier phases, occurs and enhances lignin dissolution. Finally, condensation reactions of benzylic cations with activated positions on aromatic ring were detected in lignosulfonates. This suggest that a competing reaction mode to the sulfonation at C-α position in lignin was occurring at prolonged pulping conditions, here considered to be unproductive.

Characterization of the vertical size distribution, composition and chemical properties of dissolved organic matter in the (ultra)oligotrophic Pacific Ocean through a multi-detection approach

This work presents a multi-analytical approach for the characterization of marine dissolved organic matter (DOM). The determination of marine dissolved organic carbon (DOC) was performed by size exclusion chromatography (SEC) and validated using a certified reference material (CRM) as well as through an intercomparison exercise. Multi-detection SEC, fluorescence and electrochemical methods were used in order to describe the size distribution spectra, the composition and chemical properties of marine DOM, in the (ultra)oligotrophic West Tropical South Pacific Ocean (WTSP). In this work, we defined the state of degradation of DOC in the different size fractions, operationally defined by SEC. We estimated that on average 62% of DOC was of humic nature (0.5–10 kDa), of which ~9% was able to complex trace elements, such as iron (Fe). Our results tend to support that non-refractory DOC is of high molecular weight (HMW), nitrogen (N)-rich, aliphatic, and has a weak fluorescence quantum yield and an enhanced binding capacity for Fe. The ageing of marine DOM occurring within mesopelagic waters is mainly driven by microbial respiration and alters these chemical properties. Although our results are in agreement with a paradigm describing oceanic DOM biogeochemistry known as the size-reactivity continuum, 3 μmolC L−1 of very HMW (> 10 kDa) were still observed in a water mass mainly composed of Pacific Deep Waters. This persistence could be explained by a significant content (5%) of aromatic carbon that may protect HMW DOM from long term biodegradation.

SEC coupled with in-line multiple detectors for the characterization of an oncolytic Coxsackievirus

V937 is an oncolytic virus immunotherapy clinical drug candidate consisting of a proprietary formulation of Coxsackievirus A21 (CVA21). V937 specifically binds to and lyses cells with over-expressed ICAM-1 receptors in a range of tumor cell types and is currently in phase I and II clinical trials. Infectious V937 particles consist of a ∼30 nm icosahedral capsid assembled from four structural viral proteins that encapsidate a viral RNA genome. Rapid and robust analytical methods to quantify and characterize CVA21 virus particles are important to support the process development, regulatory requirements, and validation of new manufacturing platforms. Herein, we describe a size-exclusion chromatography (SEC) method that was developed to characterize the V937 drug substance and process intermediates. Using a 4-in-1 combination of multi-detectors (UV, refractive index, dynamic and static light scattering), we demonstrate the use of SEC for the quantification of the virus particle count, the determination of virus size (molecular weight and hydrodynamic diameter), and the characterization of virus purity by assessing empty-to-full capsid ratios. Through a SEC analysis of stressed V937 samples, we propose CVA21 thermal degradation pathways that result in genome release and particle aggregation.

Comparison of Three Complementary Analytical Techniques for the Evaluation of the Biosimilar Comparability of a Monoclonal Antibody and an Fc-Fusion Protein.

In this work, a monoclonal antibody, adalimumab, and an Fc-fusion protein, etanercept, were studied and compared to one of their biosimilars. Samples submitted to stress conditions (agitation and high temperature) were used for method development. The developed methods were also applied to samples reduced by beta-mercaptoethanol to evaluate their capability to distinguish the expected species. Capillary gel electrophoresis (CGE), reversed-phase liquid chromatography (RPLC), and size-exclusion chromatography (SEC) methods coupled with UV detection were used to analyze the biopharmaceuticals. Their complementarity was investigated. For further molecular weight determination, SEC-multi angle light scattering and RPLC-quadrupole time-of-flight were occasionally used. For adalimumab, a larger amount of fragments and aggregates was observed in the biosimilar compared with the reference product. For etanercept, more related species were found in the reference product. Those three separation techniques showed good complementarity. Indeed, RPLC enabled the separation of hydrophilic and hydrophobic degradation products. CGE provided good selectivity for several adalimumab fragments, and SEC was useful for the analysis of aggregates and certain fragments that cannot be separated by the other approaches. Moreover, those formulations were submitted to mild stress conditions (30°C, 300 rpm for 4 h) that mimic shipping conditions. No additional peak was found under these conditions for the two studied biopharmaceuticals.

Assessment of the effectiveness of liquid hot water and steam explosion pretreatments of fast-growing poplar (Populus trichocarpa) wood

In this paper, the influence of physicochemical pretreatment methods on the chemical composition, enzymatic hydrolysis efficiency and porosity of fast-growing Populus trichocarpa wood was compared. Among the pretreatment methods, the liquid hot water (LHW) and steam explosion (SE) were used, which were performed at three different temperatures (160 °C, 175 °C and 190 °C) and two residence times (15 min and 1 h). The chemical composition, enzymatic hydrolysis and porosity analysis were done for native wood and solid fraction obtained after LHW and SE pretreatments. The porosity analysis was performed by inverse size exclusion chromatography method. Additionally, inhibitors of hydrolysis and fermentation processes in the liquid and solid fractions obtained after pretreatments were examined. Based on the results, it was found that the tested pretreatments caused the greatest changes in the chemical content of hemicelluloses. It was found that after LHW and SE pretreatments up to 99.1% or 94.0%, respectively, of hemicelluloses were removed from the obtained solid fraction. Moreover, the LHW and SE processes greatly enhanced the enzymatic digestibility of fast-growing poplar wood. The highest glucose yield was achieved after 15 min of SE pretreatment at 190 °C and was 676.4 mg/g pretreated biomass, while in the case of xylose the highest value (119.3 mg/g pretreated biomass) was obtained after 15 min of LHW pretreatment at 160 °C. Generally, after SE pretreatment process, more inhibitors were formed, and a greater effect of porous structure development was noticed than after LHW pretreatment. Despite this difference, the average glucose contents and yields after enzymatic hydrolysis of pretreated biomass were generally similar regardless of the pretreatment used.

Characterizing extracellular vesicles from cerebrospinal fluid by a novel size exclusion chromatography method

AbstractBackgroundExtracellular vesicles (EVs) have recently been considered as a potential biomarker source for a variety of diseases, including neurodegenerative disorders. EVs are important mediators of intercellular communication due to their capacity to transfer genetic material, lipids and proteins. By means of their communication role, interesting biomarkers are often enriched in EVs compared to total biofluid. In order to isolate EVs from a biofluid, like cerebrospinal fluid (CSF), a variety of isolation methods are available of which each methods shows different results, and, to date, there is no golden standard. Our study evaluates the use of size exclusion chromatography (SEC) by the SmartSEC HT kit for EV isolation from CSF.MethodEVs were obtained from 500µL CSF samples, isolated by SmartSEC HT. SmartSEC traps contaminants, like bulk proteins, into the resin beads and subsequently elutes EVs. To evaluate this isolation method, various characterisation analyses were run. To detect cargo or surface proteïns, analyses on the ExoView R100, Surface Plasmon Resonance imaging (SPRi) and LC‐MS based proteomics analysis were performed. Concentration, and size of the vesicles were evaluated by use of transmission electron microscopy (TEM), and fluorescence nanotracking analysis (Zetaview).ResultBy means of the ExoView, tetraspanins such as CD81 and CD9 were measured on the surface of the EVs. Other EV‐specific proteins, e.g. lactadherin, flotillin‐1, and HSP70, were detected by SPRI. Untargeted protein analysis revealed the cargo proteins. TEM images visualized the presence of EVs. Particle size and concentration was calculated by nanotracking analysis, and more specifically the particles with a lipid membrane, like EVs, with fluorescence nanotracking analysis.ConclusionAs EVs hold a lot of promise in the biomarker field, a standard high‐performance isolation method ‐ regarding purity and yield – is essential. From our data, SmartSEC HT is suggested to be an efficient size exclusion chromatography EV isolation method for low volume CSF samples, especially for high throughput, since samples are loaded on a 96‐well plate.

A simple and cost-effective synthesis of sulfated β-cyclodextrin and its application as chiral mobile phase additive in the separation of cloperastine enantiomers

A simple and cost-effective method for the synthesis of sulfated β-cyclodextrin, one of the most widely used chiral mobile phase additives, using sulfamic acid as a sulfonating agent has been described. The method was optimized, and the synthesized product was characterized by spectroscopic, size-exclusion chromatographic, thermal, and microscopic methods and was compared to the marketed Sigma Aldrich sulfated β-cyclodextrin. β-Cyclodextrin, hydroxypropyl β-cyclodextrin, sulfated β-cyclodextrin (marketed and synthesized) were evaluated as chiral mobile phase additives for the enantiomeric separation of cloperastine, an antitussive agent, using reversed-phase HPLC. Under optimized conditions, a resolution of 3.14 was achieved within 15 min on an achiral Kromasil C8 (150 × 4.6 mm, 5 µm) column, with 5 mM monopotassium phosphate containing 10 mM synthesized sulfated β-cyclodextrin pH 3.0 and 45% methanol as mobile phase. The method utilizing synthesized sulfated β-cyclodextrin as chiral mobile phase additive was validated as per ICH guidelines and applied for the quantitative determination of cloperastine enantiomers in active pharmaceutical ingredients and pharmaceutical formulations. The selectivity changes imparted by sulfated β-cyclodextrin were proven to be beneficial for chiral separation. For the enantiomeric separation of cloperastine, synthesized sulfated β-cyclodextrin afforded better resolution than marketed sulfated β-cyclodextrin.

Analysis of pharmaceutical drug oligomers by selective comprehensive two-dimensional liquid chromatography-high resolution mass spectrometry

The presence of oligomeric impurities in drugs is often overlooked and less studied due to conventional small molecule analytical methods which are often not capable of capturing these oligomers. In the present work, the oligomer species of a lipophilic active pharmaceutical ingredient (API) containing an azetidine ring was investigated. No separation was observed by reversed-phase liquid chromatography (RPLC) methods, and only a partial separation of oligomer peaks was achieved by a size exclusion chromatography (SEC) method. To improve the resolution of the different oligomers species and understand the root cause of the formation of the oligomers, a selective comprehensive two-dimensional liquid chromatography (2D-LC) method was developed by coupling SEC to RPLC. The selective comprehensive SEC × RPLC method allowed the separation of 16 species and evidenced four main groups of oligomer impurities. The contour plots of 3 API lots helped to visualize the oligomer profiles and quickly compare the difference between these lots. Finally, the oligomer peaks separated by 2D-LC were identified by high-resolution mass spectrometry (HRMS) using a Q Exactive mass spectrometer. The developed 2D-LC/HRMS workflow provides a fast and generic screening approach to quickly examine and visualize the oligomeric impurities in API materials, and direct the impurity control strategy during process development.

Identification of amylase activity from vannamei shrimps’ (Litopenaeus vannamei) digestive tract using size exclusion chromatography method

Vannamei shrimps (Litopenaeus vannamei), also known as white leg shrimps are widely cultivated and consumed by many people. However, most consumers have removed the shrimp’s head which integrates with its digestive tract. The digestive tract of white leg shrimp contains digestive enzymes, including amylase. This study aimed to determine the protein content and amylase activity from Vannamei shrimps’ digestive tract using the size exclusion chromatography method. The protein isolation of size exclusion chromatography was prepared in three steps, namely; centrifugation, precipitation, and dialysis. The protein from the dialysis step was purified by using gel filtration chromatography. Each obtained fraction was determined the protein content and amylase activity by using spectrophotometer UV-Vis method. The results showed that the fraction of 108th had the highest protein content and amylase activity with a value of 0.85 mg/ml and 25.66U/ml respectively. @font-face {font-family:"Cambria Math"; panose-1:2 4 5 3 5 4 6 3 2 4; mso-font-charset:0; mso-generic-font-family:roman; mso-font-pitch:variable; mso-font-signature:-536869121 1107305727 33554432 0 415 0;}p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-unhide:no; mso-style-qformat:yes; mso-style-parent:""; margin:0cm; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Times New Roman",serif; mso-fareast-font-family:"Times New Roman"; mso-ansi-language:EN-US;}.MsoChpDefault {mso-style-type:export-only; mso-default-props:yes; font-size:10.0pt; mso-ansi-font-size:10.0pt; mso-bidi-font-size:10.0pt; mso-ansi-language:IN; mso-fareast-language:IN;}div.WordSection1 {page:WordSection1;}

Simultaneous Analysis of Mono‐, Di‐, Triacylglycerols, and Fatty Acid Methyl Esters in Biodiesel by Size‐Exclusion Chromatography

AbstractThe purpose of this study is to develop and validate a method based on size‐exclusion chromatography (SEC) for the simultaneous determination of fatty acid methyl esters (FAME), monoacylglycerols (MAG), diacylglycerols (DAG), and triacylglycerols (TAG) in biodiesel. The proposed method presents good linearity. The limits of detection are 0.26% mass for FAME, 0.02% mass for MAG, 0.01% mass for DAG, and 0.02% mass for TAG. The limits of quantification are 0.78% mass for FAME, 0.06% mass for MAG, 0.01% mass for DAG, and 0.06% mass for TAG. Accuracy evaluated by recovery yielded values ranging from 98.93% to 117.67%. Precision is evaluated by repeatability (%), which is ranged from 0.03% to 13.67%. The proposed SEC method proves effective in determining the FAME, MAG, DAG, and TAG content of standard samples, and the paired t‐test shows that the results obtained were statistically similar to the gas chromatography (GC) values. The method also has some advantages over the reference GC methods, since it obtains the content for each class analyzed, irrespective of its components. Also, it does not require derivatization, which makes it easier and also quicker (15 min) than the 60 min taken by the two reference methods, and it does not need an internal standard, which makes it cheaper.Practical Applications: Size exclusion chromatography (SEC) is an efficient method for simultaneous and quantitative determination of fatty acid methyl esters (FAME), monoacylglycerols (MAG), diacylglycerols and triacylglycerols (TAG). The method present itself as an alternative to reference methods (ASTM D 6584 and ABNT NBR 15764) based on gas chromatography (GC). The proposed method shows advantages compared to reference methods, once it makes possible to determine the content of each constituent class in samples, regardless of its components, what makes the peak integration easier. Beyond that, previous sample derivatization is unnecessary, what makes the method simpler, cheaper and faster (15 min) than both reference methods that demands together 60 min for analysis (ASTM D 6584 for MAG, DAGa and TAG and ABNT NBR 15764 for FAME analysis).

Multi-dimensional plug-and-play liquid chromatography-native ion mobility mass spectrometry method for the analysis of biotherapeutics

A flexible inline size exclusion chromatography (SEC) and ion-exchange chromatography (IEX) method coupled to native mass spectrometry (nMS) method was developed for the analysis of protein aggregates and charge variants in a single run of biotherapeutics. This method can be applied to a broad spectrum of biologics such as human growth hormone and its soluble aggregates, the oligomer state distribution of liraglutide in solutions with different pH, and the native states of homodimers and heterodimers generated during a force stressed study of mixtures of two monoclonal antibodies. Tuning of both orbitrap and time of flight (TOF) mass spectrometers in native MS mode to accommodate this LC system is shown in this work for the analysis of biopharmaceutics. This SEC-IEX -UV-nMS system offers two-dimensional separation and two detection methods (ultraviolet absorbance and mass detection), providing an orthogonal characterization of complicated protein samples in a single method.

Isolation and characterization of urine microvesicles from prostate cancer patients: different approaches, different visions

BackgroundBecause of their specific and biologically relevant cargo, urine extracellular vesicles (EVs) constitute a valuable source of potential non-invasive biomarkers that could support the clinical decision-making to improve the management of prostate cancer (PCa) patients. Different EV isolation methods differ in terms of complexity and yield, conditioning, as consequence, the analytical result.MethodsThe aim of this study was to compare three different isolation methods for urine EVs: ultracentrifugation (UC), size exclusion chromatography (SEC), and a commercial kit (Exolute® Urine Kit). Urine samples were collected from 6 PCa patients and 4 healthy donors. After filtered through 0.22 µm filters, urine was divided in 3 equal volumes to perform EVs isolation with each of the three approaches. Isolated EVs were characterized by spectrophotometric protein quantification, nanoparticle tracking analysis, transmission electron microscopy, AlphaScreen Technology, and whole miRNA Transcriptome.ResultsOur results showed that UC and SEC provided better results in terms of EVs yield and purity than Exolute®, non-significant differences were observed in terms of EV-size. Interestingly, luminescent AlphaScreen assay demonstrated a significant enrichment of CD9 and CD63 positive microvesicles in SEC and UC methods compared with Exolute®. This heterogeneity was also demonstrated in terms of miRNA content indicating that the best correlation was observed between UC and SEC.ConclusionsOur study highlights the importance of standardizing the urine EV isolation methods to guaranty the analytical reproducibility necessary for their implementation in a clinical setting.

Validation of an Analytical Method for the Simultaneous Determination of Hyaluronic Acid Concentration and Molecular Weight by Size-Exclusion Chromatography.

The hyaluronic acid (HA) global market growth can be attributed to its use in medical, cosmetic, and pharmaceutical applications; thus, it is important to have validated, analytical methods to ensure confidence and security of its use (and to save time and resources). In this work, a size-exclusion chromatography method (HPLC-SEC) was validated to determine the concentration and molecular distribution of HA simultaneously. Analytical curves were developed for concentration and molecular weight in the ranges of 100–1000 mg/L and 0.011–2.200 MDa, respectively. The HPLC-SEC method showed repeatability and reproducibility greater than 98% and limits of detection and quantification of 12 and 42 mg/L, respectively, and was successfully applied to the analysis of HA from a bacterial culture, as well as cosmetic, and pharmaceutical products.

Establishment of a simplified dichotomic size-exclusion chromatography for isolating extracellular vesicles toward clinical applications.

Size‐exclusion chromatography (SEC) is a widely adopted method for the isolation of extracellular vesicles (EVs) from complex samples. SEC can efficiently remove high‐abundant proteins, while often requires multiple fractionation operation using diversified column settings. In this study, we aim to establish a simplified SEC method to acquire high quality EVs. In comparison of all three cross‐linked Sepharose resins with the sample types of FBS and human serum (HS), CL‐6B and CL‐4B showed superior performance in regular SEC to CL‐2B in terms of significantly narrower EV and protein peaks, higher resolutions and EV purity. By increasing their bed volumes to 20 ml, the resolutions of CL‐6B and CL‐4B columns could be significantly improved, while the CL‐6B column had the best performance with higher particle yields and tighter EV peaks. With the CL‐6B 20 ml column, we further established a simplified dichotomic SEC method that only requires two bulk elutions to acquire EVs in the Eluate 1 and proteins in the Eluate 2. We further justified that such CL‐6B columns were reusable for at least 10 consecutive times, and the dichotomic SEC was applicable to EV isolations from HS and FBS‐free supernatants of fluorescently labelled and unlabelled SW620 cells. The proteomics analysis implicated that although the two methods had dissimilar abilities in removing different co‐isolating contaminant proteins from EVs, the dichotomic SEC and ultracentrifugation could isolate EVs from human plasma with comparable purity. This dichotomic SEC has its intriguing potential to be used for EV preparation toward clinical testing and/or basic research.

Size exclusion chromatography of aromatic compounds in high-boiling petroleum samples

Knowledge of aromatic compounds in petroleum samples is of paramount interest from processing and environmental viewpoints. Considering the complexity of such samples, a selective analytical strategy viz. aromatic specific size exclusion chromatographic methodology using amino-bonded silica phase, was adopted to obtain the average molecular weights of distilled cuts, residue and the corresponding crude oil. The method was found to be suitable from middle distillates up to the residue. It was found that the increase in boiling temperature of distilled cuts was reflected in an increase in high molecular weight aromatic species. The increase in the molecular weight of aromatic species is linked to the increase in alkylation as verified by studying the elution pattern of alkylated aromatic compounds. The accuracy of the molecular weights obtained from the size exclusion chromatographic method depends on the elution profile of aromatic species in petroleum samples. It was found that the size and shape of the parent aromatic compounds negatively influence the size exclusion elution profile. However, such influence can be assumed to be negligible as all the aromatic species of each distilled cut, residue, and crude oil eluted in the permeation zone of the chromatogram. This method has the potential to be used in the second dimension of a 2D-LC method where the aromatic compounds are separated based on the size (or alkylation). The first dimension will separate compounds based on the number of aromatic rings, thereby eliminating the interference of parent aromatic compounds of different sizes in the total elution profile.

Uncertainty of Size-Exclusion Chromatography Method in Quality Control of Bevacizumab Batches

In addition to the analytical challenges related to the size and complexity of biopharmaceutical drugs, the inherent variability that arises due to their manufacturing process requires monitoring throughout the production process to ensure the safety and efficacy of the finished product. In this step, validation data should demonstrate that the process is controlled and reproducible, whereas the manufacturing process must ensure the quality and consistency of the product. For this, the manufacturer sets specification limits according with regulatory guidance. In such a situation, the comparison of different batches is required in order to describe and analyze the variability between them. However, it is unclear how great the variability of the analytical method would be or that in producing the batches. The estimation of the β-expectation tolerance intervals based on the variance components to account for both between-batch and within-batch variability was proposed as a specification limit to control the heterogeneity between batches at the time of manufacture and to verify whether batches meet specification limits. At this point, the variance components were computed by the maximum likelihood method using a linear random model. For this, the protein content, expressed as a percentage of the actual concentration relative to the claim value, and the dimer content (expressed as percentage) were used as critical quality attributes (CQAs) in the monitoring and control process. We used real data from six bevacizumab commercial batches.

Identification of Tomato Infecting Viruses That Co-Isolate with Nanovesicles Using a Combined Proteomics and Electron-Microscopic Approach.

Plant-derived nanovesicles (NVs) have attracted interest due to their anti-inflammatory, anticancer and antioxidative properties and their efficient uptake by human intestinal epithelial cells. Previously we showed that tomato (Solanum lycopersicum L.) fruit is one of the interesting plant resources from which NVs can be obtained at a high yield. In the course of the isolation of NVs from different batches of tomatoes, using the established differential ultracentrifugation or size-exclusion chromatography methods, we occasionally observed the co-isolation of viral particles. Density gradient ultracentrifugation (gUC), using sucrose or iodixanol gradient materials, turned out to be efficient in the separation of NVs from the viral particles. We applied cryogenic transmission electron microscopy (cryo-TEM), scanning electron microscopy (SEM) for the morphological assessment and LC–MS/MS-based proteomics for the protein identification of the gradient fractions. Cryo-TEM showed that a low-density gUC fraction was enriched in membrane-enclosed NVs, while the high-density fractions were rich in rod-shaped objects. Mass spectrometry–based proteomic analysis identified capsid proteins of tomato brown rugose fruit virus, tomato mosaic virus and tomato mottle mosaic virus. In another batch of tomatoes, we isolated tomato spotted wilt virus, potato virus Y and southern tomato virus in the vesicle sample. Our results show the frequent co-isolation of plant viruses with NVs and the utility of the combination of cryo-TEM, SEM and proteomics in the detection of possible viral contamination.

Functional and structural properties of gum arabic complexes with casein and hydrolyzed casein achieved by Maillard reaction

In this study, casein was extracted from milk and hydrolyzed with pepsin at three different enzyme/substrate ratios (0.1:100, 0.15:100, and 0.2:100). Three molar ratios of casein or hydrolyzed caseing/gum Arabic (GA) (1:1, 1:2, and 2:1) were exposed to Millard reaction using wet heating method at 80 °C, 6 h. The conjugation of biopolymers through Millard reaction was verified by Fourier Transform Infrared (FT-IR), high performance size exclusion chromatography (HPSEC) and fluorescence spectrophotometry methods. Higher molecular weight was observed after the reaction experiment. Moreover, FT-IR spectrum showed changes in amide 1 and 2 bands and fluorescence spectrophotometry showed reduction of intrinsic fluorescence intensity. Maillard reaction got faster by increasing the degree of hydrolyzation and GA/hydrolyzed casein ratio. Conjugates with higher ratios of GA/casein showed more stable particle size distribution against freeze-thawing process. Increasing the degree of hydrolyzation did not affect significantly on particle size distribution of conjugates. Functional properties of conjugated biopolymers including solubility, emulsifying and foaming properties were promoted compared to casein and casein/GA mixture. Also, hydrolyzed casein-GA showed better emulsifying and foaming ability but less foaming stability than non-hydrolyzed samples.

Analyzing liver protein-bound DMAV by using size exclusion and ion exchange HPLC combined with ICP-MS and MRM mode in rats exposed to AS4S4

Long-term exposure to high levels of arsenic (As) will result in damage to organs. Compared with free arsenic, protein-bound arsenic are more difficult to be excreted from the bodies due to their complexation with biological macromolecules. We developed a method of size exclusion chromatography (SEC) and ion exchange chromatography (IEC) combined with inductively coupled plasma-mass spectrometry (ICP-MS) and multiple reaction monitoring (MRM) mode, which was used to determine bound-arsenic species. DMAV was identified as bound arsenic species in rat livers after As4S4 overexposure. Subsequent proteomics analysis showed the potential binding partners included hemoglobin, glutathione S-transferases, superoxide dismutase [Cu–Zn] & [Mn], thiosulfate sulfurtransferase, and metallothionein-2. The method developed here was sensitive, repeatable, and conducive to arsenic analysis, especially for toxicity evaluation of arsenic-containing substances in vivo.

Stability-Indicating Analytical Approach for Stability Evaluation of Lactoferrin.

Lactoferrin is a multifunctional iron-binding glycoprotein in milk. Due to its potential for the treatment of various diseases, interest in products containing lactoferrin is increasing. However, as a protein, it is prone to degradation, which critically affects the quality of products. Therefore, the main purpose of our work was to develop a stability-indicating analytical approach for stability evaluation of lactoferrin. We were focused on two complementary methods: reversed-phase and size-exclusion chromatography. The stability-indicating nature of the selected methods was confirmed. They were successfully validated by following the ICH guidelines and applied to preliminary lactoferrin stability studies. Up to three degradation products, as well as aggregates and fragments of lactoferrin, were detected in various samples using complementary reversed-phase and size-exclusion chromatographic methods. The analytical approach was additionally extended with three spectroscopic techniques (absorbance, intrinsic fluorescence, and bicinchoninic acid method), which may provide valuable complementary information in some cases. The presented analytical approach allows the stability evaluation of lactoferrin in various samples, including the ability to detect differences in its degradation mechanisms. Furthermore, it has the potential to be used for the quality control of products containing lactoferrin.