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.

Synthetic approach to iodosulfuron-methyl and metsulfuron-methyl metabolites and their application for water analysis.

A synthetic approach to ten metabolites of iodosulfuron-methyl sodium and metsulfuron-methyl was performed and reported in this study. The compounds of interest were prepared by controlled hydrolytic degradation of active substances or by de novo synthesis from commercially available triazine precursor 10. Obtained compounds were characterized by IR, NMR, and elemental analysis techniques. Metabolites and active substances were utilized during the development of a separation and quantification method using reversed-phase high-performance liquid chromatography coupled with tandem mass spectrometry. The validated method was applied for the analysis of all studied compounds in the extracts from water samples collected from the Vistula river (Torun, Poland).

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.

Hydrophilic interaction liquid chromatography with mass spectrometry for the separation and identification of antisense oligonucleotides impurities and nusinersen metabolites.

With the development of therapeutic oligonucleotides for antisense and gene therapies, the demand for analytical methods also increases. For the analysis of complex samples, for example plasma samples, where the use of mass detection is essential, hydrophilic interaction liquid chromatography is a suitable choice. The aim of the present work was to develop a method for separation and identification of the oligonucleotide impurities and metabolites by hydrophilic interaction liquid chromatography. First of all, the effects of different chromatographic conditions (e.g. pH of the aqueous part of the mobile phase, buffer concentration, column temperature) on the retention and separation of phosphorothioate oligonucleotides standards on the amide stationary phase were investigated. A set of model oligonucleotides containing a fully modified 21mer and its typical impurities (shortmers and oligonucleotides with different number of thiophosphate modifications) was used. The results showed that the concentration of the salt in the mobile phase as well as its pH, are the most influential parameters with regard to peak shape and separation. The knowledge gained was applied to the analysis of an unpurified 18mer oligonucleotides, analogues of the drug nusinersen used for the treatment of spinal muscular atrophy. The successful separation and identification of twenty-six and twenty-eight impurities was performed with the developed HILIC method. The method was applied to analysis of nusinersen metabolites of serum samples of patients treated with Spinraza.

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.

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.

Polybutylene terephthalate-based stationary phase for ion-pair-free reversed-phase liquid chromatography of small interfering RNA. Part 2: Use for selective comprehensive two-dimensional liquid chromatography.

With the increasing numbers of nucleic acid-based pharmaceuticals like antisense oligonucleotides (ASO), small interfering ribonucleic acid (siRNA) entering the market, research facilities, pharmaceutical industries and also regulatory authorities have been looking for efficient analytical methods for these synthetic oligonucleotides (ON). Besides of conventional one-dimensional (1D) reversed-phase liquid chromatography with or without ion-pairing (IP-RP-LC, RP-LC), hydrophilic liquid chromatography (HILIC) and mixed-mode chromatography (MMC), two-dimensional (2D) approaches combining two orthogonal chromatographic techniques also become more relevant due to the high structural complexity of oligonucleotides. Recently, we tested a polybutylene terephthalate(PBT)-based stationary phase under ion-pairing free RP mode for the liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS) analysis of siRNA (Patisiran). In this study, retention profile and chromatographic orthogonality, respectively, were compared to other LC-modes like HILIC, IP-RPLC, another ion-pair free cholesterol-bonded RPLC and MMC considering their normalized retention times. Finally, because of higher orthogonality, the ion-pairing free PBT-bonded RPLC as first dimension (1D) was hyphenated with HILIC in the second dimension (2D) in a selective comprehensive 2D-LC setup leading to an enhanced resolution for peak purity evaluation of the main ON entities.

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.

Polybutylene terephthalate-based stationary phase for ion-pair-free reversed-phase liquid chromatography of small interfering RNA. Part 1: Direct coupling with mass spectrometry.

Nowadays, ion-pairing reversed-phase liquid chromatography (IP-RPLC) is the dominating generic method for the analysis of nucleic acid related compounds, such as antisense-oligonucleotides (ASO), small-interfering ribonucleic acid (siRNA) or other DNA or RNA type molecules and their conjugates. Despite of its effective performance, the usage of a high concentration of ion-pairing reagent in the eluent in IP-RPLC is unfavorable for the hyphenation with mass spectrometry (MS) which is required for a detailed structural characterization of the analytes and their structurally related impurities. In this work, we tested a polybutylene terephthalate (PBT)-bonded silica-based stationary phase for the separation of generically synthesized Patisiran as siRNA (antisense and sense single strands as well as their annealed double strand) giving some unexpected selectivity without any presence of ion-pairing reagents. Important chromatographic conditions affecting the separation have been investigated and evaluated. Furthermore, MS and tandem MS (MS/MS) characterization was possible without contamination of the MS system with ion-pair agent and related problems.

A simple and green solid phase extraction method for oligonucleotides using adsorbent with amino and carboxylic moieties.

The study aimed to use a material with amino and carboxylic moieties to extract unmodified and phosphorothioate oligonucleotides. The charge of amine and carboxyl groups at the surface has changed with the change in pH of the adsorption (pH 4.5) and desorption solution (pH 9.5). Thus, both the binding and elution of the oligonucleotides were based on electrostatic interactions, and the procedure required only 10 mM ammonium acetate, with the change of pH depending on the extraction step. The developed procedure was successfully applied to extract oligonucleotides from aqueous solutions and serum samples. The method is simple and fast, providing good reproducibility (SD between 1 and 4%) and relatively high oligonucleotide recovery (81-98% for standards, 60-71% for diluted serum samples, and 80-92 for LLE serum extracts). Moreover, only environmentally friendly solvents were used.

Cholesterol Stationary Phase in the Separation and Identification of siRNA Impurities by Two-Dimensional Liquid Chromatography-Mass Spectrometry.

The aim of this research was to develop a simple and efficient ion-pair reagent-free chromatographic method for the separation and qualitative determination of oligonucleotide impurities, exemplified by synthesis of raw products of the two single strands of patisiran siRNA. The stationary phases with mixed hydrophobic/hydrophilic properties (cholesterol and alkylamide) were firstly used for this purpose with reversed-phased high-performance liquid chromatography. Several different chromatographic parameters were tested for their impact on impurities separation: type, concentration, pH of salt, as well as organic solvent type in the mobile phase. The pH was the most influential factor on the separation and signal intensities in mass spectrometry detection. Finally, the optimized method included the application of cholesterol stationary phase, with mobile phase containing 20 mM ammonium formate (pH 6.5) and methanol. It allowed good separation and the identification of most impurities within 25 min. Since not all closely related impurities could be fully resolved from the main peak in this oligonucleotide impurity profiling, two-dimensional liquid chromatography was used for peak purity determination of the target oligonucleotides. The Ethylene Bridged Hybrid (BEH) Amide column in hydrophilic interaction liquid chromatography was applied in the second dimension, allowing additional separation of three closely related impurities.

Oligonucleotides Isolation and Separation-A Review on Adsorbent Selection.

Oligonucleotides have many important applications, including as primers in polymerase chain reactions and probes for DNA sequencing. They are proposed as a diagnostic and prognostic tool for various diseases and therapeutics in antisense therapy. Accordingly, it is necessary to develop liquid chromatography and solid phase extraction methods to separate oligonucleotides and isolate them from biological samples. Many reviews have been written about the determination of these compounds using the separation technique or sample preparation for their isolation. However, presumably, there are no articles that critically review the adsorbents used in liquid chromatography or solid phase extraction. The present publication reviews the literature from the last twenty years related to supports (silica, polymers, magnetic nanoparticles) and their modifications. The discussed issues concern reversed phase (alkyl, aromatic, cholesterol, mixed ligands), ion-exchange (strong and weak ones), polar (silica, polyhydroxy, amide, zwitterionic), and oligonucleotide-based adsorbents.

Development of the Method for Nusinersen and Its Metabolites Identification in the Serum Samples of Children Treated with Spinraza for Spinal Muscular Atrophy.

The application of oligonucleotides as drugs for different genetic diseases is increasing rapidly. Since 2016 they are used during spinal muscular atrophy treatment with the use of nusinersen oligonucleotide. The purpose of this study was to improve methods for the analysis of serum samples of patients treated with nusinersen. The results showed that liquid-liquid extraction (with phenol/chloroform) is insufficient and an additional purification step using solid-phase extraction is necessary. The best results were obtained for microextraction by packed sorbents. Important parameters in the optimization of the method were mainly the type of amine in the mobile phase and the stationary phase. Both influenced the selectivity of metabolite separation and thus their correct identification; while amine type impacted also the intensity of signals. Finally, the highest resolution of separation and the highest peak areas were obtained for N,N-dimethylbutylamine or N,N-diisopropylthylamine with an octadecyl column with a terminal aryl group. Over a dozen of metabolites were successfully identified with the use of methods developed during the study. The 3' exonucleases and 5' exonucleases were mainly responsible for nusinersen metabolism, consequently, 3'end shortmers, and 5'end shortmers were observed, as well as metabolites with simultaneous loss of bases at both ends of the sequence. However, some depurination and depyrimidination products were also identified. To the best of our knowledge, this is the first report on nusinersen and its metabolite identification in serum samples by liquid chromatography and mass spectrometry.

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.

Application of Magnetic Nanoparticles Coated with Crosslinked Zwitterionic Poly(ionic liquid)s for the Extraction of Oligonucleotides.

Magnetic nanoparticles coated with zwitterionic poly(ionic liquid)s were applied for dispersive solid-phase extraction of oligonucleotides. The materials were synthesized by miniemulsion copolymerization of ionic liquids and divinylbenzene on magnetic nanoparticles. The functional monomers contain a positively charged imidazolium ring and one of the anionic groups: derivatives of acetate, malonate, or butyl sulfonate ions. Adsorption of unmodified DNA oligonucleotide on obtained materials was possible in ion-exchange (IE) and hydrophilic interactions (HI) mode. The adsorption in IE was possible at low pH and was almost complete. The adsorption in HI mode required the usage of appropriate addition of organic solvent but did not provide full adsorption. Studies on the desorption of the analytes included determining the impact of ammonium acetate concentration and pH and organic solvents addition on the recovery. The material containing acetic fragments as an anionic group was selected for the final procedure with the use of 10 mM ammonium acetate (pH = 9.5)/methanol (50/50, v/v) as an elution solution. The magnetic dispersive solid-phase extraction procedure was tested for the oligonucleotides with various modifications and lengths. Moreover, it was applied to extract DNA oligonucleotide and its synthetic metabolites from enriched human plasma without any pre-purification, with recoveries greater than 80%.

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.

Poly(ionic liquid)s as new adsorbents in dispersive micro-solid-phase extraction of unmodified and modified oligonucleotides.

Cross-linked poly(ionic liquid)s were successfully used for the first time in the preparation of oligonucleotide biological samples. The adsorbents were prepared by co-polymerization of imidazolium-based ionic liquids and divinylbenzene. Consequently, the following three adsorbents were prepared and comprehenzively characterized: poly(3-butyl-1-vinylimidazolium bromide-co-divinylbenzene), poly(3-hexyl-1-vinylimidazolium bromide-co-divinylbenzene) and poly(2-(1-vinylimidazoliumyl)acetate-co-divinylbenzene). Oligonucleotides were adsorbed onto the surface of these materials at low pH values. Preliminary studies of the desorption of the analytes included testing the influence of different types of salts, as well as their concentrations and pH, and organic solvents on the recovery. This allowed for determining the adsorbent and the desorption conditions for further optimization with the use of central composition design. The chosen adsorbent was poly(2-(1-vinylimidazoliumyl)acetate-co-divinylbenzene), and the optimal desorption conditions (5 mM ammonium acetate (pH = 9.5)/methanol (50/50, v/v)) gave a recovery of 99.7 ± 0.3%. The dispersive micro-solid-phase extraction procedure was successfully applied for the extraction of oligonucleotides with various modifications and lengths. Finally, the developed method was used to extract 2'-O-methyl oligonucleotide and its two synthetic metabolites from enriched human plasma without any pre-purification, yielding recoveries over 80%.

Studying in vitro metabolism of the first and second generation of antisense oligonucleotides with the use of ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry.

The aim of the present investigation was the analysis and identification of antisense oligonucleotide metabolism products after incubation with human liver microsomes regarding four different oligonucleotide modifications. Separation and detection methods based on the use of liquid chromatography coupled with quadrupole time-of-flight mass spectrometry were developed for this purpose. Firstly, the optimization of mass spectrometer parameters was done to select those which ensure the highest possible sensitivity of oligonucleotide analysis. This step was conducted for two chromatographic modes-ion pair chromatography and hydrophilic interaction liquid chromatography-due to their common application in oligonucleotide analysis. Based on sensitivity results, ion pair chromatography coupled with mass spectrometry was selected for the separation of model oligonucleotide mixtures in order to verify its selectivity for N-deleted metabolite separation. Next, the developed method was applied in the examination of oligonucleotides in vitro metabolism. First, wide optimization of incubation parameters was conducted including the concentration of the reaction buffer components. Obtained results indicated that both 3'-exonucleases and 5'-exonucleases contributed to the biotransformation of oligonucleotides. Moreover, it may be concluded that the number of metabolites depends on oligonucleotide modification and consequently its resistance to enzymatic attack. Thus, the number of the oligonucleotide metabolites decreased with the decrease of the resultant polarity of oligonucleotide caused by chemical modification. Graphical abstract.

Application of hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry for the retention and sensitivity studies of antisense oligonucleotides.

The aim of the present investigation was application of hydrophilic interaction liquid chromatography as an alternative chromatographic approach for the study of antisense oligonucleotides. The influence of several mobile phases, differing with the salt type, their concentration and pH value on the retention and the separation of antisense oligonucleotides has been examined for this purpose. Four different stationary phases were also applied including unmodified silica, silica modified with the use of sulfobetaine groups, polyhydroxy and aminopropyl groups. Such wide range of tested conditions has been useful in better understanding of the retention mechanism of tested compounds. The results obtained during this investigation indicated that greater retention, greater peaks symmetry, as well as more effective separation of oligonucleotides, were obtained for the zwitterionic stationary phase. Moreover, the optimization of tandem mass spectrometry parameters with the use of Central Composite Design was performed and different mobile phases were tested to choose that one, which provided the greatest antisense oligonucleotides peak areas in Multiple Reaction Monitoring mode and consequently, the greatest possible sensitivity. Hydrophilic interaction liquid chromatography was compared with the ion pair chromatography, commonly used in the analysis of oligonucleotides. Both techniques were compared in terms of selectivity of separation as well as the sensitivity of their determination. Obtained results proved that ion pair chromatography provided better results in terms of separation efficiency and peak areas in Multiple Reaction Monitoring for tested conditions. However, these results do not preclude application of hydrophilic interaction liquid chromatography as an alternative chromatographic approach for the oligonucleotides analysis especially when a mobile phase without ion pair reagents is required.