Study of nusinersen metabolites in the cerebrospinal fluid of children with spinal muscular atrophy using ultra-high-performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry.

The study aimed to analyze nusinersen metabolites in the cerebrospinal fluid samples using ion-pair reversed-phase ultrahigh-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. Three different sample preparation methods were tested for extraction and purification, but solid phase extraction appeared to be the most suitable, allowing a significant sample enrichment (40-fold). This step was necessary to detect and identify metabolites of nusinersen in the cerebrospinal fluid. The developed and applied analytical procedure enabled the identification of nusinersen metabolites: sequences shorter by several nucleotides from the 3' end; shorter by several nucleotides from both the 3' and 5' ends; and some depurination products. To the best of our knowledge, this is the first report on the analysis and identification of nusinersen metabolites in cerebrospinal fluid samples taken from children with spinal muscular atrophy treated with Spinraza.

Exploring the green frontier: Subcritical water chromatography for sustainable analytical practices.

Water in the subcritical state is characterized by properties significantly different from water under standard conditions. These include low viscosity, low surface tension, and a much lower dielectric constant, increasing the solubility of nonpolar substances. For this reason, it can provide an alternative solvent and be used in chromatographic techniques-subcritical water chromatography (SBWC). SBWC appears to be one of the greenest analytical techniques until we unravel chromatography with pure water at room temperature. The versatility of SBWC is explored through its applications in the separation and analysis of a wide range of compounds, including pharmaceuticals, natural products, etc. The use of subcritical water as a mobile phase requires suitable stable stationary phases and special apparatus. Still, it makes it possible to conduct analyses without using organic solvents. When using this technique, it is important to remember that it suits the analysis of thermally stable substances. The following work is a critical review of developments in SBWC.

Elimination of Toxic Solvents from Analytical Methods in Food Analysis: Caffeine Determination in Tea as an Example.

This study presents an innovative method for caffeine determination in tea, employing ethanol as the sole organic solvent for both SPE sample preparation and chromatographic analysis. This approach aligns with green chemistry principles, as confirmed by a comparative study highlighting ethanol's safety and eco-friendliness compared to traditional solvents. The experiments validate ethanol's efficacy in caffeine extraction and chromatographic analysis, minimizing environmental impact and eliminating toxicity risks. Utilizing a reduced chromatography column enhances the method's efficiency and sustainability, resulting in a low limit of quantitation (0.125 µg/mL) and good reproducibility (RSD < 2.5%). Based on tea from the Polish market, the findings reveal the caffeine content (19.29-37.69 mg/g) and endorse ethanol's role in enhancing sustainable chemical analysis in food science.

Retention mechanism on phosphodiester stationary phases in hydrophilic interaction liquid chromatography and purely aqueous mobile phase part II: Overloading with limited soluble samples.

The adsorption behaviour of caffeine and theophylline under hydrophilic interaction chromatography and purely aqueous conditions was investigated on four phosphodiester stationary phases. Solute adsorption isotherms were determined by frontal analysis or inverse method. The bi-Langmuir model was found to be the best choice to describe the behaviour of caffeine and theophylline adsorption in purely aqueous conditions, whereas the bi-Moreau model describes the adsorption phenomena in HILIC conditions. The results obtained demonstrate that the interaction of caffeine and theophylline with the stationary phase surface varies depending on the mobile phase composition. Both in pure aqueous mobile phase and in HILIC mode, the heterogeneity of the surface of the studied stationary phases is confirmed. In hydrophilic solutions, the sample molecules interact with the stationary phase only. In hydrophobic conditions, a lateral interaction occurs between caffeine or theophylline molecules, which are poorly soluble in acetonitrile-rich solvents. This confirms that the same compound on the same stationary phase can behave rather differently, depending on the mobile phase composition. Thus, the mobile phase may govern and control the retention mechanism.

Extraction of Nucleotides from Dietary Supplements by Newly Synthesized Adsorbents.

The aim of the study was the synthesis and application of novel adsorbents for the extraction of nucleotides from dietary supplements. Three different adsorbents modified with a silane containing two amine groups and various dicarboxylic acids were synthesized and characterized using various instrumental techniques. Next, different solvents were tested for their adsorption and desorption of five nucleotides. The results showed that the efficiency of both processes depends on the conditions used and the type of dicarboxylic acid bound to the surface of the adsorbent. The best results were obtained for succinic acid. The most effective adsorption occurred for water acidified with acetic acid to pH 4.5, while the highest recoveries (85-102%) with high reproducibility were obtained for 10 mM ammonium acetate at pH 9. The nucleotide extraction was performed simply by changing the charge at the adsorbent surface, providing the possibility of electrostatic attraction and repulsion between the adsorbent and nucleotides. Moreover, the sorption capacity of the obtained materials was also determined, which was essential for their use in extracting nucleotides from real samples by dispersive extrusion to the solid phase. The new adsorbents and the developed extraction method were successfully applied to isolate nucleotides from two different dietary supplements with different compositions (one of them with yeast strains). The method is simple and reproducible; no organic solvents or high-concentration inorganic salts are used (it is environmentally friendly). The entire process is performed in one centrifuge tube and is cheaper compared with methods used so far.

Fabrication and characterization of new levan@CBD biocomposite sponges as potential materials in natural, non-toxic wound dressing applications.

Wound healing is a complex process; therefore, new dressings are frequently required to facilitate it. In this study, porous bacterial levan-based sponges containing cannabis oil (Lev@CBDs) were prepared and fully characterized. The sponges exhibited a suitable swelling ratio, proper water vapor transmission rate, sufficient thermal stability, desired mechanical properties, and good antioxidant and anti-inflammatory properties. The obtained Lev@CBD materials were evaluated in terms of their interaction with proteins, human serum albumin and fibrinogen, of which fibrinogen revealed the highest binding effect. Moreover, the obtained biomaterials exhibited antibacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa, as well as being non-hemolytic material as indicated by hemolysis tests. Furthermore, the sponges were non-toxic and compatible with L929 mouse fibroblasts and HDF cells. Most significantly, the levan sponge with the highest content of cannabis oil, in comparison to others, retained its non-hemolytic, anti-inflammatory, and antimicrobial properties after prolonged storage in a climate chamber at a constant temperature and relative humidity. The designed sponges have conclusively proven their beneficial physicochemical properties and, at the preliminary stage, biocompatibility as well, and therefore can be considered a promising material for wound dressings in future in vivo applications.

A Determination of the Caffeine Content in Dietary Supplements According to Green Chemistry Principles.

Caffeine is a natural psychoactive substance that belongs to a group of chemical compounds called purine alkaloids. Caffeine is found in various plants such as coffee, tea, cocoa, guarana, and yerba mate. It is often added to dietary supplements for its ability to increase metabolism and aid in weight loss. To determine the caffeine content in dietary supplements, a novel UHPLC method was developed, compatible with the rules of green analytical chemistry. The developed method used only water and ethanol for sample preparation and chromatographic separation on a short C18 column. The obtained method confirmed that caffeine may be analyzed using only environmentally friendly solvents, ethanol, and water. The developed method is characterized by its low limit of quantitation, equal to 0.047 µg/mL, and good reproducibility (a relative standard deviation lower than 1.1%). The obtained results show that the caffeine content in tested dietary supplements is 4-35% higher than the declared amount in most cases. In comparison, the caffeine content of the drug determined using this method was performed with an accuracy of 0.4% RSD.

Phosphodiester Stationary Phases as Universal Chromatographic Materials for Separation in RP LC, HILIC, and Pure Aqueous Mobile Phase.

Modern analytical chemistry techniques meet the need for greater attention to ecological and economic aspects. It is becoming necessary to seek solutions to reduce harmful waste production, especially in large quantities. High-performance liquid chromatography is a technique widely used in many industries, including mainly pharmaceuticals, and requires an approach to reduce the significant amount of organic solvent waste. One of the green chemistry solutions is using environmentally benign substitutes, such as pure water, supercritical dioxide, and ethanol. Our work focuses on the preparation and application of new stationary phases with embedded hydrophilic groups for separations using pure water in liquid chromatography. Polar-embedded stationary phases are obtained by attaching a phosphodiester group and 4 different hydrophobic molecules. The studies consisted of hydrophobicity measurements, concentration dependence of retention of the organic additive to the mobile phase, and chromatographic separations of polar and non-polar substance mixtures in RP-LC and HILIC systems. Three mixtures were studied: purine alkaloids, benzene, and polycyclic aromatic hydrocarbons and nucleosides. The stationary phases interact differently with the analytes depending on the attached hydrophobic group. It is possible to use pure water to separate each mixture under study. It is also significant that it has been possible to separate a mixture of completely non-polar compounds using pure water for the first time. The research being carried out is crucial in synthesizing new polar-embedded stationary phases, providing work versatility and high environmental performance.

Beta-Blocker Separation on Phosphodiester Stationary Phases-The Application of Intelligent Peak Deconvolution Analysis.

Beta-blockers are a class of medications predominantly used to manage abnormal heart rhythms. They are also widely used to treat high blood pressure. From the liquid chromatography separation point of view, beta-blockers are interesting molecules due to their hydrophobic-hydrophilic properties. Thus, the study aimed to investigate the beta-blocker separation selectivity on four phosphodiester stationary phases in reversed-phase liquid chromatography (RP LC) and hydrophilic interactions liquid chromatography (HILIC). On tested stationary phases, beta-blockers provide retention in both chromatographic systems, RP LC and HILIC. Additionally, it was found that cation-exchange mechanisms have a significant contribution to retention. Separations were enhanced by applying ChromSword software for gradient optimization and Intelligent Peak Deconvolution Analysis to separate unseparated peaks digitally.

Chitosan-based films with cannabis oil as a base material for wound dressing application.

This study focuses on obtaining and characterizing novel chitosan-based biomaterials containing cannabis oil to potentially promote wound healing. The primary active substance in cannabis oil is the non-psychoactive cannabidiol, which has many beneficial properties. In this study, three chitosan-based films containing different concentrations of cannabis oil were prepared. As the amount of oil increased, the obtained biomaterials became rougher as tested by atomic force microscopy. Such rough surfaces promote protein adsorption, confirmed by experiments assessing the interaction between human albumin with the obtained materials. Increased oil concentration also improved the films' mechanical parameters, swelling capacity, and hydrophilic properties, which were checked by the wetting angle measurement. On the other hand, higher oil content resulted in decreased water vapour permeability, which is essential in wound dressing. Furthermore, the prepared films were subjected to an acute toxicity test using a Microtox. Significantly, the film's increased cannabis oil content enhanced the antimicrobial effect against A. fischeri for films in direct contact with bacteria. More importantly, cell culture studies revealed that the obtained materials are biocompatible and, therefore, they might be potential candidates for application in wound dressing materials.

Optimization of the packing process of microcolumns with the embedded phosphodiester stationary phases.

The development of new home-made stationary phases involves their packaging procedure and is crucial to obtain satisfactory working parameters. The parameter that illustrates the quality of the packed bed is its efficiency measured as the height equivalent to the theoretical plate. According to the Van Deemetr's equation, it depends on three factors, but only one of them, eddy diffusion, does not depend on the linear flow velocity. Therefore, in order to obtain it as low as possible, it is necessary to focus on a good filling of the column. Among many parameters affecting the quality of column packing, in our work we have focused on the choice of slurry solvent. Novel stationary phases with an embedded phosphodiester group were investigated. The suspensions in 16 solvents and solvent mixtures were studied for their stability, aggregation, sedimentation, and viscosity comparison. The efficiency of the packed microcolumns and its comparison was determined by chromatographic analyses using a polar (thymidine) and a nonpolar compound (naphthalene). The results obtained led to the conclusion that for these stationary phases, the best slurry solvent is the one that aggregates the phase while maintaining stability and having high viscosity.

Dendrimer Anion-Exchange Stationary Phase for Separation of Oligonucleotides.

Oligonucleotides are used in many research areas. Thus, there is a need for their successful separation methods. Ion-exchange chromatography is the most popular separation technique, but it has limitations for these compounds. For this reason, new stationary phases are developed in order to increase separation selectivity. This study aimed to apply a series of dendrimer anion exchangers with various bonded layers to separate oligonucleotides by using different mobile phases to find conditions that allow sufficient separation. The number of anion-exchange layers, type of salt, and pH significantly impacted the oligonucleotide analysis. The developed chromatographic method was characterized by adequate selectivity for oligonucleotides differing in sequence length. It is essential to underline that the number of bonded layers appeared to have a significant influence, and the three layers appeared optimal. Based on our results, it may be concluded that the dendrimer stationary phases can be successfully used as an alternative to commonly applied packing materials in ion-exchange chromatography.

Solvent Influence on Zeta Potential of Stationary Phase-Mobile Phase Interface.

Zeta potential is a surface characteristic formed on the solid surface and liquid interface. It is an interesting way to describe the surface properties of materials; thus, a series of four homemade polar embedded stationary phases that contain phosphate groups incorporated into hydrophobic ligands were investigated according to surface zeta potential. Measurements were carried out using Zetasizer Nano ZS for the stationary phases suspensions prepared in various solvent and solvent binary mixtures. The negative zeta potential values were obtained for most cases due to negatively charged residual silanols and phosphate groups. However, in some solvents: tetrahydrofuran, isopropanol, and toluene zeta potential are positive. Additionally, it was observed that the zeta potential seems to be independent of the type of silica gel used for the stationary phase synthesis.

Stationary Phases for Green Liquid Chromatography.

Industrial research, including pharmaceutical research, is increasingly using liquid chromatography techniques. This involves the production of large quantities of hazardous and toxic organic waste. Therefore, it is essential at this point to focus interest on solutions proposed by so-called "green chemistry". One such solution is the search for new methods or the use of new materials that will reduce waste. One of the most promising ideas is to perform chromatographic separation using pure water, without organic solvents, as a mobile phase. Such an approach requires novel stationary phases or specific chromatographic conditions, such as an elevated separation temperature. The following review paper aims to gather information on stationary phases used for separation under purely aqueous conditions at various temperatures.

Photochemical Properties and Stability of BODIPY Dyes.

The present study is devoted to the combined experimental and theoretical description of the photophysical properties and photodegradation of the new boron-dipyrromethene (BODIPY) derivatives obtained recently for biomedical applications, such as bacteria photoinactivation (Piskorz et al., Dyes and Pigments 2020, 178, 108322). Absorption and emission spectra for a wide group of solvents of different properties for the analyzed BODIPY derivatives were investigated in order to verify their suitability for photopharmacological applications. Additionally, the photostability of the analyzed systems were thoroughly determined. The exposition to the UV light was found first to cause the decrease in the most intensive absorption band and the appearance of the hypsochromically shifted band of similar intensity. On the basis of the chromatographic and computational study, this effect was assigned to the detachment of the iodine atoms from the BODIPY core. After longer exposition to UV light, photodegradation occurred, leading to the disappearance of the intensive absorption bands and the emergence of small intensity signals in the strongly blue-shifted range of the spectrum. Since the most intensive bands in original dyes are ascribed to the molecular core bearing the BF2 moiety, this result can be attributed to the significant cleavage of the BF2 ring. In order to fully characterize the obtained molecules, the comprehensive computational chemistry study was performed. The influence of the intermolecular interactions for their absorption in solution was analyzed. The theoretical data entirely support the experimental outcomes.

Synthesis and application of stationary phase for DNA-affinity chromatographic analysis of unmodified and antisense oligonucleotide.

The goal of the research was the synthesis and application of an oligonucleotide immobilized stationary phase for the analysis of unmodified and antisense oligonucleotides. The method for attaching these molecules to aminopropyl silica modified with pentanedioic acid was developed. Each step of the synthesis was carefully controlled with the application of spectroscopic, elemental, and chromatographic analyses. The oligonucleotide-based stationary phase was applied for the retention studies. Unmodified oligonucleotides of different complementarity to the molecule attached as a stationary phase, as well as antisense oligonucleotides, were tested. The comparative study upon complex optimization of oligonucleotide analysis in different liquid chromatography modes was performed. Results have shown that this stationary phase may be applied for oligonucleotide analysis in hydrophilic interaction liquid chromatography and ion exchange chromatography, but no unique sequence-based selectivity was obtained. Contrary results were observed for affinity chromatography, which allowed for specific separation of the complementary strands based on hydrogen bonding and stacking interactions, where the temperature was the main factor influencing the selectivity of the separation. Furthermore, the oligonucleotide-based stationary phase may be applied for comparative antisense oligonucleotide hybridization studies to a specific RNA sequence. All of the results have shown that affinity chromatography with oligonucleotide-based stationary phases is a powerful technique for the specific base recognition of polynucleotides.

Surface properties of stationary phases with embedded polar group based on secondary interaction, zeta potential measurement and linear solvatation energy relationship studies.

The chromatographic properties of six non-commercially available stationary phases with ester or phosphodiester functional groups embedded into alkyl chain were studied. Zeta potential values of stationary phases suspended in water, organic solvent and their mixtures were measured. Moreover, the selectivity coefficients were calculated on the basis of the retention factor for the test solutes. Separations were performed under hydrophilic interaction liquid chromatographic conditions. Hydrophobic and polar properties of the investigated columns were compared. Based on the chromatographic properties, polar embedded packing materials were classified. Also two phases with different spacer but the same embedded polar group and alkyl chain (Amino-P-C18 and Diol-P-C18) were used for comparison with homemade materials. Amino-P-C18 stationary phase exhibits positive values of zeta potential which is in accordance with the observed anion exchange properties. The highest negative values of zeta potential were observed for Diol-P-C18, together with cation exchange properties. The highest methylene selectivity, polar selectivity and configurational selectivity were observed for the stationary phase with an ester bond and a phenyl group.

Attachment of hybridizable oligonucleotides to a silica support and its application for selective extraction of unmodified and antisense oligonucleotides from serum samples.

The main aim of the present study was the synthesis of an oligonucleotide-based material with high chemical stability, repeatability and specificity to complementary oligonucleotides. The oligonucleotides were attached to a silica gel surface modified with amino acids during one-step synthesis. The amount of the oligonucleotides immobilized on the support surface had an impact on adsorption effectiveness, due to steric interference. The adsorption capacity corresponds to 4.7 µg of complementary oligonucleotide per 1 mg of material, which reflects 50% of immobilized oligonucleotides. The presented results contain comprehensive studies on hybridization and release of fully complementary, partially complementary, non-complementary and antisense oligonucleotides from the newly synthesized adsorbent. The salt concentration and time period were the most influential parameters in the case of adsorption, while high temperature and low salt content were indispensable for effective desorption. Selectivity studies revealed that the adsorption percentage increases with the decreasing number of base mismatches. Consequently, the desorption of low complementarity oligonucleotides was always greater in comparison with the fully complementary sequence. Furthermore, it was shown that oligonucleotide-based materials may be successfully used for the extraction of antisense oligonucleotides and their metabolites from serum samples with recoveries ranging between 65 and 73%.

Quantitative structure - retention relationships of amino acids on the amino acid- and peptide-silica stationary phases for liquid chromatography.

Quantitative structure - retention relationships analysis was applied to investigate the molecular retention mechanism of proteinogenic and non-proteinogenic amino acids on the amino acid- and peptide-silica stationary phases. Twelve stationary phases with chemically bonded amino acids of different types (glycine, alanine, phenylalanine, leucine, methionine, aspartic acid, and N-(9-Fluorenylmethoxycarbonyl)-O-tert-butyl-l-tyrosine) and chains lengths (amino acid, dipeptide, and tripeptide) were tested. In order to compare chromatographic properties of the prepared materials with the conventional columns, the amino-bonded phases (laboratory-prepared and commercial one) were also studied. For each of analyte, the molecular descriptors were calculated using quantum mechanics method. The QSRR models were determined using 13 molecular descriptors mainly related to the surface area, hydrophobicity, polarity, ion-exchange and hydrogen bonding capabilities of the analytes. Finally, the prediction potency of the molecular modeling descriptors-based models was also independently studied for the tested stationary phases using 15 training set and 6 test set of amino acids.