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.

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.

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.

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.

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.

The versatility of N,O-dialkylphosphoramidate stationary phase-separations in HILIC, highly aqueous RP LC conditions and purely aqueous mobile phase.

The chromatographic properties of N,O-dialkylphosphoramidate stationary phase with an octadecyl chain (Amino-P-C18) were studied. Based on the U-shape retention plots over the mobile phase composition for the particular tested compounds (nucleosides and nucleic bases) and solvent excess adsorption isotherms, the separations of the tested compounds were performed in various chromatographic conditions. The separations were performed via hydrophilic interaction liquid chromatography, in highly aqueous mobile phase as well as using pure water as a mobile phase. The obtained results confirm that the specific structure of the amino-P-C18 stationary phase allows solvation over a whole range of mobile phase compositions. The ability of this material to undergo chromatographic separation in extreme mobile phase compositions (hydrophilic interaction chromatography and purely aqueous systems) was also confirmed. Successful separation of test mixtures using pure water as a mobile phase is in accordance with the rules of "green chemistry".

The competitiveness of solvent adsorption on polar-embedded stationary phases.

Solvation processes on polar embedded stationary phases were investigated by determination of excess isotherms in binary acetonitrile/water mobile phase. The obtained results indicate the competitiveness of acetonitrile and water adsorption on heterogeneous surfaces of chemically bonded stationary phases. The calculation of excess isotherm as a volume of excessively accumulated solvent confirms the competitiveness of solvents' adsorption in qualitative and quantitative meaning. Corresponding excess isotherms of water and acetonitrile are symmetrical with respect to the x-axis. It confirms that the displaced volume of acetonitrile is replaced by an equal volume of water and vice versa. Additionally, fitting the two-site adsorption model to experimental data provides quantitative values with low experimental errors. The obtained results confirm the advantage of volumetric assumption of excess adsorption over direct adsorption layers of solvents, which is commonly postulated.

Correction: A study of separation selectivity using embedded ester-bonded stationary phases for liquid chromatography.

Correction for 'A study of separation selectivity using embedded ester-bonded stationary phases for liquid chromatography' by Szymon Bocian et al., Analyst, 2016, 141, 4340-4348.

The effect of solvation processes on amino acid- and peptide-silica stationary phases.

The surface excess adsorption isotherms of water, acetonitrile, and methanol from binary hydro-organic mobile phases were investigated on nine home-made stationary phases with chemically bonded amino acids, dipeptides, and tripeptides using the dynamic minor disturbance method. The stationary phases were modified by the following amino acids: glycine, alanine, phenylalanine, leucine, and aspartic acid. We investigated the influence of the type of immobilized amino acids, in particular their different side chains, on the solvent adsorption. The interpretation of solvation phenomena shows significant accumulation of investigated solvents on the adsorbent surface according to their hydrophilic or hydrophobic properties. Moreover, the accumulated amount was dependent on the length and type of amino acid sequences bonded to the silica surface. Stationary phases with bonded amino acids and peptides show stronger water and acetonitrile adsorption in contrast to the stationary phase modified with aminopropyl groups-a support for the synthesis. The comparison of water and acetonitrile adsorption as well as a data obtained with the two-site adsorption model reveal and confirm the heterogeneity of chemically bonded phases. As a consequence of performed investigations, the classification of tested stationary phases for the potential usage in particular high-performance liquid chromatography mode was also accomplished.

A study of separation selectivity using embedded ester-bonded stationary phases for liquid chromatography.

A new type of stationary bonded phase for liquid chromatography with various functional groups bonded to diol-modified silica via ester bond was synthesized. The structures of the proposed stationary phases contain: alkyl chains (C10, C18), phenyl, and cholesterol groups. The structures of the synthesized materials were confirmed by different physico-chemical techniques such as elemental analysis, infrared spectroscopy (FTIR), (13)C CP/MAS NMR and liquid chromatography under reversed phase conditions (RP) and with hydrophilic interaction liquid chromatography (HILIC). Depending on the type of functionalities bonded to the Diol-Ester, the stationary phases are capable of separating various groups of compounds in RP and HILC, even using pure water as a mobile phase.

Amino acids, peptides, and proteins as chemically bonded stationary phases--A review.

The selectivity of chromatographic separation depends mostly on the stationary phase and mobile phase composition. Despite being a material with bonded simple organic molecule, a wide group of stationary phases contain immobilized compound that possesses biological activity. Stationary phases that contain amino acids and peptides as well as enzymes and proteins are alternative materials that may be used for liquid chromatographic separations and are reviewed in this work. In the case of peptide-bonded stationary phases, most of these types of materials were elaborated in the 1970s and 1980s; however, over the last few years a growing interest has been observed which is connected with hydrophilic interaction liquid chromatography. The most important application of amino acid and peptide-bonded stationary phases is connected with separation of amino acids, their derivatives, and peptides. The main advantage of such materials is the ability for chiral separations.

Solvation processes on phenyl-bonded stationary phases-The influence of polar functional groups.

A series of phenyl-bonded stationary phases with incorporated polar functional groups was subjected to an adsorption investigation. Measurement of acetonitrile and methanol adsorption was obtained using the minor disturbance method. It was observed that adsorption of organic solvent strongly depends on the presence of polar functional groups in the bonded phases that influence the hydrophobicity and polarity of the stationary phase surface. Additionally, relative adsorption of acetonitrile and methanol confirms earlier observations, that the presence of amine and amide groups in the stationary phase changes the relative elution strength of organic solvents. The heterogeneous surface of the stationary phase makes it possible to observe the competitiveness of the water and organic solvent adsorption.

Influence of the charge distribution on the stationary phases zeta potential.

A set of seven home-made silica based bonded phases with different functional groups was investigated. Their zeta potential data in methanol and acetonitrile as well as in methanol/water and acetonitrile/water solution were obtained by using a Zetasizer. The influence of polar functional groups on a zeta potential was investigated. The results show that the amines incorporated in the structure of chemically bonded phases of reversed-phase materials are protonated during chromatographic analysis, resulting in changes of the zeta potential from negative to positive values. Acetonitrile causes more negative values and methanol provides positive (or less negative) values of the zeta potential.

Thermodynamics Study of Solvent Adsorption on Octadecyl-Modified Silica.

Elution and solvation processes in liquid chromatography may be controlled by temperature changes. In the case of solvent adsorption, the temperature influences the amount of adsorbed solvent as well as the enthalpy and entropy of the solvation process. In this work, the thermodynamic parameters of organic solvents used as organic modifiers in the reversed-phase high-performance liquid chromatography elution process were determined. The changes of enthalpy and entropy in a series of chemically bonded stationary phases were measured to determine the effects of the temperature and surface coverage density of octadecyl ligands on the thermodynamic parameters of the solvation. For both the enthalpy and entropy a parabolic trend was observed with the minimum for medium surface coverage. The correlation of solvent adsorption values with the enthalpy of solvation was also investigated. The highest influence of the temperature on solvation process was observed for stationary phases with high surface coverage.

Phenyl-bonded stationary phases--the influence of polar functional groups on retention and selectivity in reversed-phase liquid chromatography.

The chromatographic properties of four phenyl-bonded phases with different structures were studied. The columns used were packed with a stationary phase containing a phenyl ring attached to the silica surface using different types of linkage molecules. As a basic characteristic of the bonded phases, the hydrophobicity and silanol activity (polarity) were investigated. The presence of the polar amino and amide groups in the structure of the bonded ligand strongly influences the polarity of the bonded phase. Columns were compared according to methylene selectivity using a series of benzene homologues and according to their shape and size selectivity using polycyclic aromatic hydrocarbons. The measurements were done using methanol/water and acetonitrile/water mobile phases. The presented results show that the presence of polar functional groups in the ligand structure strongly influences the chromatographic properties of the bonded phase.

Comparison of four cholesterol-based stationary phases for the separation of steroid hormones.

The chromatographic behavior of steroid hormones on four cholesterol-bonded stationary phases with different structures in binary methanol/water mobile phases was studied. Of the stationary phases tested, the commercially available stationary phases Cogent UDC cholesterol™ and COSMOSIL cholester™ provided better separations of steroid hormones in comparison to homemade aminocholesterol and diaminocholesterol stationary phases. The results show that the temperature has a significant influence on the retention and selectivity for steroid hormones separation. The temperature increase may cause changes in the elution order. From the dependences of the retention (ln k) on temperature (1/T), the standard partial molar enthalpy and standard partial molar entropy were calculated and their enthalpic and entropic contributions to the retention were compared. The enthalpic effects principally control the retention mechanism.