Electrophoresis Method Research Articles

Offline Coupling of Hydrophobic Interaction Chromatography-Capillary Zone Electrophoresis for Monitoring Charge-Based Heterogeneity of Recombinant Monoclonal Antibodies.

A holistic understanding of the charge heterogeneity in monoclonal antibodies (mAbs) is paramount for ensuring acceptable product quality. Hence, biotherapeutic manufacturers are expected to thoroughly characterize their products via advanced analytical techniques. Recently, two-dimensional liquid chromatography (2DLC) methods have gained popularity for resolving complex charged species. Capillary electrophoresis (CE) is regarded as a sensitive and faster tool for charged species estimation in biotherapeutics. In this study, we aim to combine the separation power of chromatographic and electrophoretic tools (liquid chromatography [LC]-CE) so as to achieve maximum resolution of mAb charge variants. Hydrophobic interaction chromatography (HIC) has been used as the preferred LC mode with CE for achieving successful separation of both charge and hydrophobic variants for two of the mAbs (trastuzumab and rituximab). The standalone HIC and capillary zone electrophoresis (CZE) methods separated 4 hydrophobic variants and 7 charge variants for each mAb, whereas the 2DLC method separated 10 and 11 variants for mAbs A and B. On the other hand, the HIC-CZE-UV method resolved 29 variants in mAb A and 23 variants in mAb B. The reproducibility of the HIC-CZE-UV method was demonstrated by % change in values of retention time (RT) and peak area as <5% (mAb A), <3% (mAb B), and <12% (for both mAbs), respectively. Thus, the utility of the proposed LC-CE method for characterization of mAb charge variants has been displayed.

Determination of an Anti-Parasitic Active Pharmaceutical Ingredient in Wastewater Effluents Using Capillary Zone Electrophoresis.

Ireland has a successful pharmaceutical industry with over 100 pharmaceutical manufacturing sites across the island. Although this success has many benefits, the irreversible effects emissions from pharmaceutical manufacturing can have on the environment are a major drawback. Although known pollutants are regularly monitored with limits set out by the Environmental Protection Agency, one significant pollutant has been overlooked: pharmaceutical pollution. Detecting these pollutants and ensuring they are at a safe concentration for the environment is of utmost importance. In recent years, capillary electrophoresis is being recognised as a suitable alternative to high-performance liquid chromatography due to its many benefits such as faster analysis, water-based buffers and smaller sample volumes. In this paper, a capillary zone electrophoresis (CZE) method with a preconcentration step of solid-phase extraction was developed for an anti-parasitic active pharmaceutical ingredient (API) called ZB23. The API was successfully detected in a wastewater sample in less than 10min using the CZE parameters of 25mM borate buffer with a pH of 10.5, 15% MeOH, 10kV voltage, 25mbar for 5s injection size, an Lt of 40cm, an Ld of 31.5cm and a detection wavelength of 214nm.

The method of capillary electrophoresis for quantitative determination of hydrophobized hyaluronic acid in its micellar forms

Micelles based on hydrophobized hyaluronic acid (HA) are frequently used in targeted drug delivery systems. Capillary zone electrophoresis (CZE) was utilized for the quantitative determination of hydrophobized and native HA. A universal methodology was developed, suitable for the quantitative analysis of amphiphilic derivatives of hyaluronan (oleyl hyaluronan and hyaluronan conjugate with naphthalimide fluorophore) and native HA with varying molecular weights (15, 150, and 800 kDa). Furthermore, methodologies were proposed for the simultaneous quantification of a drug substance and oleyl hyaluronan in micellar forms based on the latter. The CE technique was applied for analyzing oleyl-hyaluronan-based micellar forms of two poorly soluble drug substances with oppositely charged ionic forms (loperamide and rifabutin). The examples contained in the study demonstrate a range of analytical sensitivity (LOD) for hyaluronan from 11 to 40 μg/mL and for the drug substance from 0.4 to 0.6 μg/mL. The study also showcases the accurate quantitative determination of rifabutin and loperamide in oleyl-hyaluronan-based micellar forms without the need for sample preparation. Thus, the proposed methodologies can be used to quantify native HA or its amphiphilic derivatives and simultaneously determine drug substances of various nature.

Proteomic Profile of Blood Plasma of Mus Musculus in the Acute Toxicity Test of Single Black Garlic (Allium Sativum)

This study aims to obtain overall information about all proteins formed in body fluids, cells, or tissues at a certain time and to analyze the mechanism of cell responses to various types of stress and drugs. Single black garlic (SBG) has the potential to be a pharmacological agent. However, the safety of using single black garlic is still unknown, so a material toxicity test is needed. This research is a laboratory experimental research with a post-test-only control group design. An oral acute toxicity test was used. Observation time was 14 days and consisted of four treatments, negative control, SBG dose 2000 mg/kgbw (P1), 3000 mg/kgbw (P2), and 5000 mg/kgbw (P3). Post-treatment protein analysis was done using the SDS-PAGE electrophoresis method. Observations of clinical symptoms of SBG at doses of 2000 mg/kgbw, 3000 mg/kgbw, and 5000 mg/kgbw showed no clinical symptoms in mice, both motor activity and pupils were still normal, no convulsions (seizures), lacrimation, paralysis or death were seen in mice. Observations on plasma proteins showed differences in protein between the treatments at the SBG dose of 2000 mg/kgbw with the SBG dose of 3000 mg/kgbw and 5000 mg/kgbw, but the four treatments have the same type of protein in the protein band with a molecular weight of 25 kDa. So, it can be concluded that SBG doses of 2000 mg/kgbw, 3000 mg/kgbw, and 5000 mg/kgbw are not toxic to mice but can stimulate the expression of certain proteins. Keywords: Single Black garlic (SBG), oral acute toxicity test, proteomic analysis, SDS-PAGE

Stacking in electrophoresis by electroosmotic flow-assisted admicelle to solvent microextraction.

An in-line sample concentration method for capillary electrophoresis called admicelle to solvent microextraction was proposed. In this technique, analytes were trapped in the cetyltrimethylammonium bromide admicelles formed in situ on the negatively charged capillary surface. A solvent plug was then partially injected hydrodynamically to collapse the admicelles, which liberated and focused the analytes at the solvent front. Voltage was applied across the capillary, completing the stacking process. Various solvents, namely, methanol, ethanol, and acetonitrile, were investigated. The optimal solvent for solvent to admicelle microextraction was 30% acetonitrile in 24mM sodium tetraborate (pH 9.2). Sample injection time and solvent to sample injection ratio were also optimised. For this demonstration, the optimum sample injection time and solvent to sample injection ratio were 320s and 1:2, respectively. Using the optimum conditions, UV detection sensitivity was enhanced 132-176-fold for the model anions. The LOQ, %intra-/inter-day (n = 6/n = 12, 2days) repeatability, and linearity (R2) of admicelle to solvent microextraction were 0.08-2µg/mL, 1.9-3.9%, 2.8-4.9%, and 0.992, respectively. Admicelle to solvent microextraction was applied to the analysis of various fortified water samples, with good repeatability (%RSD = 0.5-3.6%), and no matrix interferences.

Greening Separation and Purification of Proteins and Peptides.

The increasing awareness of environmental issues and the transition to green analytical chemistry (GAC) have gained popularity among academia and industry in recent years. One of the principles of GAC is the reduction and replacement of toxic solvents with more sustainable and environmentally friendly ones. This review gives an overview of the advances in applying green solvents as an alternative to the traditional organic solvents for peptide and protein purification and analysis by liquid chromatography (LC) and capillary electrophoresis (CE) methods. The feasibility of using greener LC and CE methods is demonstrated through several application examples; however, there is still plenty of room for new developments to fully realize their potential and to address existing challenges. Thanks to the tunable properties of designer solvents, such as ionic liquids and deep eutectic solvents, and almost infinite possible mixtures of components for their production, it is possible that some new designer solvents could potentially surpass the traditional harmful solvents in the future. Therefore, future research should focus mainly on developing new solvent combinations and enhancing analytical instruments to be able to effectively work with green solvents.

Выбор рационального инструментария для анализа полифенолов цветков бессмертника песчаного и листьев артишока колючего в комплексе фармакологических исследований

Background: Pathology of the hepatobiliary system includes a heterogeneous group of diseases of the liver and biliary system caused by viral, bacterial and parasitic infections, neoplasms, toxic chemicals, including alcohol, nutritional errors, metabolic disorders and heart failure. One of the basic groups for pharmacological correction of this group of diseases is the use of hepatoprotectors, drugs that have a predominantly selective effect on the liver. The active ingredient of most plant hepatoprotectors are flavonoids and hydroxycinnamic acids. The most popular plants – hepatoprotectors containing these active substances include – artichoke prickly (Cynara scolymus (L.)) and sandy everlasting (Helichrysum arenarium (L.) Moench), a plant with choleretic action, also capable of hepatoprotective activity. The aim of the study: Development and comparative characterisation of methods for the analysis of polyphenolic composition of sandy everlasting flowers and artichoke leaves in complex pharmacological studies using HPLC and capillary electrophoresis methods. Materials and methods: The sum of polyphenolic compounds was subjected to chromatographic separation in gradient elution mode. Capillary electrophoresis was carried out in a variant of micellar electrokinetic chromatography (MEKC). The buffer solution used was a mixture of: borate buffer 20 mM (pH - 9.3) – beta-cyclodextrin 20 mM – ethyl alcohol (10:10:5). Results: The electrophoregrams of the separation of artichoke leaf extract by the MECC method showed the presence of 7 components belonging to oxycinnamic acids and flavonoids. The dominant ones are chlorogenic acid and luteolin7-glucoside. Similar results were obtained by the HPLC method. The electrophoregram of immortelle flower extract shows 9 components, of which the dominant one belongs to isosalipurposide. Chromatographic analysis of sandy everlasting flower extract showed the presence of the same components. Conclusion: The results of the analysis of the polyphenolic complex of artichoke leaves and sandy everlasting flowers by capillary electrophoresis agree with the data obtained by HPLC chromatography. This allows us to conclude that the MECC method allows the identification and quantification of each component in artichoke leaves and HPLC chromatography flowers along with HPLC. Moreover, the performance of the analysis by CE is more economically feasible than HPLC, since it does not require the use of solvents for the mobile phase and the availability of a set of columns.

Comparative analysis of protein profiles in skin secretions of some Rana species: Preliminary insights into antimicrobial activity

Protein profiles of skin secretions of Rana dalmatina (Agile Frog), Rana macrocnemis (Uludağ Frog), Rana tavasensis (Tavas Frog) and Rana holtzi (Taurus Frog) frog species belonging to the Rana genus distributed in the Anatolian region of Türkiye were determined for the first time using the Tricine-SDS-PAGE Electrophoresis method and Coomassie Brilliant Blue (CBB) staining. By the results, some peptides with mass ≤5 kDa were detected. Just one peptide with mass ≤5 kDa was found in the secretion of each R. dalmatina, R. macrocnemis, and R. tavasensis while there was two in R. holtzi secretion. The antibacterial activity of secretions was determined using plate well diffusion assay on E. coli, S. typhimurium, S. aureus, B. cereus and L. monocytogenes bacteria. R. dalmatina created the inhibition zone for S. typhimurium, S. aureus, B. cereus, and L. monocytogenes. The zones of inhibition by R. tavasensis and R. macrocnemis species secretions were observed on S. aureus, B. cereus, and L. monocytogenes. It was found that R. holtzi creates an inhibition zone only on B. cereus. The results showed that the secretion of none of the species doesn't have antibacterial activity on E. coli. The skin secretion of R. dalmatina showed the most activity against bacteria, while R. holtzi had the least.

Synthesis and Molecular Dynamic Simulation of Novel Cationic and Non-cationic Pyrimidine Derivatives as Potential G-quadruplex-ligands.

Drug resistance has been a problem in cancer chemotherapy, which often causes shortterm effectiveness. Further, the literature indicates that telomere G-quadruplex could be a promising anti-cancer target. We synthesized and characterized two new pyrimidine derivatives as ligands for G-quadruplex DNA. The interaction of novel non-cationic and cationic pyrimidine derivatives (3a, b) with G-quadruplex DNA (1k8p and 3qsc) was explored by circular dichroism (CD) and ultraviolet-visible spectroscopy and polyacrylamide gel electrophoresis (PAGE) methods. The antiproliferative activity of desired compounds was evaluated by the MTT assay. Apoptosis induction was assessed by Propidium iodide (P.I.) staining and flow cytometry. Computational molecular modeling (CMM) and molecular dynamics simulation (MD) were studied on the complexes of 1k8p and 3qsc with the compounds. The van der Waals, electrostatic, polar solvation, solventaccessible surface area (SASA), and binding energies were calculated and analyzed. The experimental results confirmed that both compounds 3a and 3b interacted with 1k8p and 3qsc and exerted cytotoxic and proapoptotic effects on cancer cells. The number of hydrogen bonds and the RMSD values increased in the presence of the ligands, indicating stronger binding and suggesting increased structural dynamics. The electrostatic contribution to binding energy was higher for the cationic pyrimidine 3b, indicating more negative binding energies. Both experimental and MD results confirmed that 3b was more prone to form a complex with DNA G-quadruplex (1k8p and 3qsc), inhibit cell growth, and induce apoptosis, compared to the non-cationic pyrimidine 3a.

Rapid identification of antibody impurities in size-based electrophoresis via CZE-MS generated spectral library

Methods for the reliable and effective detection and identification of impurities are crucial to ensure the quality and safety of biopharmaceutical products. Technical limitations constrain the accurate identification of individual impurity peaks by size-based electrophoresis separations followed by mass spectrometry. This study presents a size-based electrophoretic method for detecting and identifying impurity peaks in antibody production. A hydrogen sulfide-accelerated degradation method was employed to generate known degradation products observed in bioreactors that forms the basis for size calibration. LabChip GXII channel electrophoresis enabled the rapid (< 1 min) detection of impurity peaks based on size, while capillary zone electrophoresis-mass spectrometry (CZE-MS) facilitated their accurate identification. We combine these techniques to examine impurities resulting from cell culture harvest conditions and forced degradation to assess antibody stability. To mimic cell culture harvest conditions and the impact of forced degradation, we subjected samples to cathepsin at different pH buffers or exposed them to high pH and temperature. Our method demonstrated the feasibility and broad applicability of using a CZE-MS generated spectral library to unambiguously assign peaks in high throughput size-based electrophoresis (i.e., LabChip GXII) with identifications or likely mass of the antibody impurity. Overall, this strategy combines the utility of CZE-MS as a high-resolution separation and detection method for impurities with size-based electrophoresis methods that are typically used to detect (not identify) impurities during the discovery and development of antibody therapeutics.

Determination of the Effect of Thymoquinone on DNA Damage in Kidney Cells Treated to High Glucose Depending on Time and Dosage by Comet Assay

The purpose of this study was to assess the anti-genotoxic potential of thymoquinone (TQ) against DNA damage in NRK-52E cells treated with high glucose using the comet assay technique single cell gel electrophoresis method. Cells were propagated by regular passages in in vitro conditions. TQ proliferative concentration (10μM) and IC25 (3rd-hour: 550 mM, 12th-hour: 240 mM, 24th-hour: 200 mM) and IC50 (3rd-hour: 760 mM, 12th-hour: 400 mM, 24th-hour: 280 mM) values for each hour of high glucose and were determined separately with MTT method. At these concentrations, the cells were divided into control(C), Thymoquinone (TQ), high glucose(G) and high glucose plus thymoquinone (GT) groups; It was incubated with the indicated substances for 3, 12, 24 hours. DNA damage was evaluated by applying the comet assay protocol and the results were calculated as DNA damage index (DDI). While DDI levels were observed to be significantly higher (p&lt;0.05) in all groups administered high glucose compared to the control, a significant decrease was determined in all groups in which TQ was added along with high glucose. It was determined that high concentrations of glucose had genotoxic effects on kidney cells, and TQ administration together with high glucose, depending on concentration and time, had a significant effect on reducing DNA damage. However, it was concluded that the application of only thymoquinone significantly increased the DDI value compared to the control, and this was a data worth investigating in future studies. Additionally, TQ inhibited DNA damage. These results demonstrated the importance of TQ against nephrotic syndrome with its high antioxidant properties.

Physicochemical Characterization of the Oral Biotherapeutic Drug IMUNOR®.

IMUNOR is an oral biotherapeutic drug that had been developed, registered, and approved in 1997 in the Czech Republic and Slovakia. IMUNOR is a dialyzable leukocyte extract (DLE) prepared from swine leukocytes. It is characterized as a mixture of small peptides with molecular weights smaller than 12 kDa and a specific portion of nucleotides. The medical uses of IMUNOR include therapeutic applications within its registered range of indications, primarily for the treatment of immunodeficiencies, allergies, and certain acute or relapsing bacterial infections in adults and children. Despite the long-term clinical application of DLE, with strong evidence of positive therapeutic effects and no serious side effects, a detailed physicochemical specification of this mixture was lacking. We developed several methods for more in-depth physicochemical characterization of IMUNOR, including a spectrophotometric method for quantification of the total protein concentration and total DNA concentration in a mixture, several chromatographic methods for identification of individual components present in significant concentrations in IMUNOR, such as HPLC methods and the Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis method, and characterization of amino acid composition of this mixture. For the investigation of the variability among different batches of IMUNOR, five to nine representative batches from a standard manufacturing process on an industrial scale were utilized. Using the analytical methods, we verified and confirmed the batch-to-batch reproducibility of the biological product IMUNOR.

Effect of chitosan conjugates with oxycinnamic acids and &lt;i&gt;Bacillus subtilis&lt;/i&gt; bacteria on the activity of protective proteins and resistance of potato plants to &lt;i&gt;Phytophthora infestans&lt;/i&gt;

The effect of chitosan conjugates with caffeic (ChCA) and ferulic (ChFA) acids in combination with Bacillus subtilis bacteria on the transcriptional activity of PR protein genes and proteome changes in potato plants during infection with Phytophthora infestans (Mont.) de Bary was studied. Plants grown from mini tubers of the Luck variety were sprayed with solutions of ChCA and ChFA, suspension of B. subtilis bacteria strains 26D and 11 VM, composites of ChCA of ChFA together with bacteria. 3 days after treatment, some of the plants were infected with P. infestans. A decrease in the degree of development of the pathogen of late blight on potato leaves in all treatment options was revealed. The maximum protective effect was manifested when plants were treated with bacteria B. subtilis strain 26D in combination with conjugates of chitosan and oxycinnamic acids. The mechanisms of increasing the resistance of potato plants to P. infestans were associated with the activation of transcriptional activity of genes encoding the main protective protein (PR‑1), chitinase (PR‑3), thaumatin-like protein (PR‑5), protease inhibitor (PR‑6), peroxidase (PR‑9), ribonuclease (PR‑10). The revealed activation of the expression of marker genes of systemic acquired resistance and induced systemic resistance under the influence of joint treatment of plants with B. subtilis and chitin conjugates with oxycinnamic acids indicate the synergistic development of protective reactions in potato plants in this variant. By the method of two-dimensional electrophoresis of S. tuberosum leaf proteins followed by MALDI-TOF analysis, 12 proteins were identified, the presence of which in the leaves differed depending on the variant of the experiment. In all treatment variants, suppression of serine-threonine protein phosphatase activity was observed, reflecting the development of the hypersensitivity reaction. Different variants of the experiment formed weakly expressed clusters, which indicates multiple mechanisms of regulation of the synthesis of protective proteins involved in the reaction to treatment with bacteria, chitosan conjugates and infection with P. infestans.

SHEDDING LIGHT ON GENETIC RELATIONSHIPS, PLASMID PROFILES AND ANTIBIOTIC RESISTANCE PATTERNS OF CLINICAL ESCHERICHIA COLI STRAINS: PULSED-FIELD GEL ELECTROPHORESIS PERSPECTIVE

In the present work, 47 Escherichia coli isolates collected from various clinical samples were elucidated with regard to genetic relationship, antibiotic resistance and plasmid profile. The genetic diversity of the isolates was defined by the pulsed field gel electrophoresis (PFGE) method. With a degree of similarity of 80%, the results from PFGE separated the E. coli strains into 39 different groups representing four subtypes. Antibiotic resistance patterns and extended spectrum of beta lactamase (ESBL) producing properties of the strains were determined by using a disk diffusion and double disc synergy method, respectively. According to the susceptibility test results, 36 distinct resistance profiles (resisto type) were observed among clinical strains. The strains were mostly resistant to ampicillin (100%) and this has been followed by cephalothin and ticarcillin/clavulanic acid with the ratio of 89.36%, cefuroxime with the ratio of 87.24%, tetracycline and cefotaxime with the ratio of 80.85%. It was observed that 24 of the strains (51.06%) were defined as ESBL positive. When E. coli strains were evaluated according to plasmid size, it was determined that 27 of 47 strains (57.44%) carried plasmids and the sizes of the determined plasmids were ranging between 77.1 kb and 1.6 kb. It was concluded that plasmids of E. coli strains are randomly distributed and no significant correlation was found between antibiotic resistance patterns and plasmids.

Enhanced Commendable Sensitivity and Specificity for MSI in Colorectal Cancer by a New PCR-HRM Based 8-Loci MSI Assay.

This study evaluated the performance of the PCR-HRM assay by comparing it with immunohistochemistry (IHC) for mismatch repair (MMR) proteins and the PCR capillary electrophoresis (PCR-CE) methods. A total of 224 patients with colorectal cancer participated in the study, with nearly half having mismatch repair deficiency (dMMR) tissues and the remainder possessing pMMR tissues. There was a 97.77% concordance between the PCR-HRM assay and IHC, and a 97.56% concordance between PCR-HRM and the PCR-CE assay. In comparison with IHC for dMMR proteins, the PCR-HRM demonstrated a sensitivity of 96.36% and a specificity of 99.12%. When juxtaposed with the PCR-CE assay, its sensitivity was 98.96% and specificity stood at 96.33%. The mutations observed in the microsatellite loci were uniformly distributed across all eight loci. Discrepant outcomes were more frequent in instances of MLH1 and PMS2 deficiency. Furthermore, the germline mutation status of MLH1, MSH2, PMS2, and MSH6 in 62 patients was ascertained using next-generation sequencing. All patients displaying MMR gene pathogenic mutations (N = 14) were identified as MSI-H by PCR-HRM, whereas those with MSS tissues (N = 43) did not exhibit MMR gene pathogenic mutations. Thus, the PCR-HRM method proficiently pinpoints tumors with verified germline MMR mutations, indicative of Lynch syndrome. Conclusively, the PCR-HRM assay emerges as a swift and congruent diagnostic tool for microsatellite instability, boasting commendable sensitivity and specificity in colorectal cancer.

Assessment of DNA quality for whole genome library preparation

In recent years, more sophisticated DNA technologies for genotyping have enabled considerable progress in various fields such as clinical genetics, archaeogenetics and forensic genetics. DNA samples previously rejected as too challenging to analyze due to low amounts of degraded DNA can now provide useful information. To increase the chances of success with the new methodologies, it is crucial to know the fragment size of the template DNA molecules, and whether the DNA in a sample is mostly single or double stranded. With this knowledge, an appropriate library preparation method can be chosen, and the DNA shearing parameters of the protocol can be adjusted to the DNA fragment size in the sample. In this study, we first developed and evaluated a user-friendly fluorometry-based protocol for estimation of DNA strandedness. We also evaluated different capillary electrophoresis methods for estimation of DNA fragmentation levels. Next, we applied the developed methodologies to a broad variety of DNA samples processed with different DNA extraction protocols. Our findings show that both the applied DNA extraction method and the sample type affect the DNA strandedness and fragmentation. The established protocols and the gained knowledge will be applicable for future sequencing-based high-density SNP genotyping in various fields.

Comparative analysis of electrophoresis and interferometric optical detection method for molecular weight determination of proteins

Analytical detection methods play a pivotal role in scientific research, enabling the identification and quantification of specific analytes in various disciplines. This scientific report aims to compare two very different methodologies for determining the Molecular Mass (MM, also known as Molecular Weight, MW) of proteins: electrophoresis gel and the Interferometric Optical Detection Method (IODM). For this purpose, several proteins with different MM were selected.The electrophoresis technique was employed to validate the structure and MM of different parts or fragments of the Matrix Metallopeptidase 9 antibody (anti-MMP9), antibody against S100 calcium binding protein A6 (anti-S100A6) and Cystatin S4 antibody (anti-CST4) by examining the presence of bands with expected sizes. The IODM was applied to study the above-mentioned proteins (part of the antibodies) together with the protein G, as a reference to correlate the MM and protein sizes with the measured signal.We report the evidence of IODM as a competitive analytical approach for the determination of the MM of proteins for the first time. This innovative method allows for accurate MM determination using minimal sample volumes and concentrations, employing a simple experimental procedure that eliminates the requirement for protein denaturation.

Development of a capillary electrophoresis method with laser-induced fluorescence detection for the characterization of oligosaccharides in human milk

Breast milk is the most appropriate food for an infant in the first months of life - it provides the essential nutrients required for proper growth, with breast milk oligosaccharides (HMOs) being the third most abundant component. HMO concentrations vary based on factors like lactation stage and maternal health. They play crucial roles in infant health, acting as prebiotics, antimicrobials, and immune modulators. Analytical methods like capillary electrophoresis and liquid chromatography are the most often used in HMOs qualitative and quantitative analysis. Continuing research aims to enhance analytical techniques for comprehensive HMOs analysis, this work presents the development of a capillary electrophoresis method with laser-induced fluorescence detection along with derivatization and solid-phase extraction steps for the characterisation of major oligosaccharides in colostrum samples. Validation parameters, such as linearity (0.9949–0.9989), limits of detection (5.49–16.40 ng cm−3) and quantification (18.30–54.67 ng cm−3), intra- and inter-day precision (2.41–9.78% and 1.27—14.5%, respectively), trueness (− 8.4 to − 2.0%), and recovery (70.02–113.5%) and repeatability (2.22–10.58%) for solid-phase extraction stage were assessed. The method was evaluated using RGB additive colour model regarding the analytical and practical effectiveness and greenness of the method (achieving a brilliance score of 67.1%). Moreover, the developed method was successfully applied to determine three oligosaccharides (DSLNT, 3’SL, and 6’SL) in colostrum samples. Considering its effectiveness, the method has promise for this type of application.Graphical abstract

The use of liquid and gas chromatography in combination with mass spectroscopy and the method of capillary electrophoresis to study hydroxycinnamic acids in plants growing in Russia

BACKGROUND: Recently, the volume of research and publications devoted to the study of the class of phenylpropanoids in plants, in particular, hydroxycinnamic acids and their derivatives, has been steadily growing. This is due to the diverse range of their pharmacological activity and the possibility of using them in medicine. The reliability and reliability of identification of this group of compounds is significantly expanding with the improvement of analytical methods: gas and liquid chromatography in combination with mass spectroscopy, as well as the use of capillary electrophoresis. The study of the proposed approaches to the analysis of this group of compounds can be interpolated to new plant objects. AIM: A comparative analysis of the use of liquid and gas chromatography methods in combination with mass spectroscopy, as well as the capillary electrophoresis method for the study of hydroxycinnamic acids in plants growing and cultivated in the Russian Federation. MATERIALS AND METHODS: Results published in domestic periodical scientific journals and conference proceedings, based on information and analytical studies. RESULTS: Data on the study of the conditions of extraction, structure and content of hydroxycinnamic acids and their derivatives in plants growing on the territory of the Russian Federation have been systematized. The review presents the advantages and limitations of gas and liquid chromatography in combination with the method of mass spectroscopy in the analysis of this group of compounds, using various conditions for their extraction from plant materials. The prospects of using the capillary electrophoresis method for these purposes are shown due to the ease of implementation and highly efficient separation of hydroxycinnamic acids and their derivatives in plant materials. It has been established that there is a lack of information on studying the dynamics of their accumulation depending on climate-forming factors, regions of growth, as well as their stability in plant materials. CONCLUSION: analysis of the methods presented in the review allows us to create a methodological basis for further improvement and development of new methods for the analysis of hydroxycinnamic acids and their derivatives in plant objects.

A novel graphene-N/TiO2-nickel foam: Preparation and application in photoelectric synergistic degradation of methyl orange

Photoelectric synergistic degradation of dye wastewater has been developed. In this experiment, we prepared Nitrogen-doped titanium dioxide-graphene/nickel foam (N/TiO2-Gr/F–Ni) by electrophoresis and impregnation methods, to degrade methyl orange (MO) aqueous solution. Firstly, nitrogen-doped titanium dioxide particle (N/TiO2) was prepared by the sol-gel method, which showed a photocatalytic degradation catalytic kinetic constant of 1.8 × 10−2 min−1 under simulated sunlight. Meanwhile, graphene (Gr) was loaded on nickel foam (F–Ni) to prepare Gr/F–Ni electrode by electrophoresis method, which obtained a catalytic kinetic constant of 1.15 × 10−2 min−1 at 10 V. Finally, N/TiO2 was loaded on Gr/F–Ni to prepare N/TiO2-Gr/F–Ni photoelectrode by electrophoresis and impregnation methods, which had a catalytic kinetic constant of 3.96 × 10−2 min−1 at 10 V and under 1 sun. The results show that there is a synergistic effect between light degradation and electro-Fenton oxidation because the bonding electrons in N/TiO2 are activated to produce photogenerated electron-hole pairs. On the other hand, the separation efficiency of the photogenerated electron is enhanced by the Gr.