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A selective and simple method was developed for the determination of four ethyleneamines (EAs), i.e., ethylenediamine (EDA), diethylenetriamine (DETA), triethylenetetramine (TETA), and tetraethylenepentamine (TEPA), by reversed-phase ion-pair chromatography with ultraviolet detection. The method is based on the on-line complexation of EAs with a Cu(II) ion added to the mobile phase. The Cu(II) complexes with EAs (Cu(II)-EA complexes) were well separated on a reversed-phased column, using 1-octanesulfonate as an ion-pairing reagent. The concentrations of 1-octanesulfonate and acetonitrile in the mobile phase significantly influenced the retention times of EAs. The high concentration of the Cu(II) ion in the mobile phase resulted in the increase of the baseline noise. A mixture of acetonitrile and 50 mM acetate buffer at pH 5.5 (23:77, v/v) which contains 0.5 mM CuSO 4 and 20 mM 1-octanesulfonic acid sodium salt was used as the mobile phase. Cu(II)-EA complexes could be detected at 243 nm. The limits of quantification of EAs (S/N = 10) were 0.003 mM for EDA, DETA, and TETA and 0.01 mM for TEPA. The calibration curves were linear over three orders of magnitude of EA concentrations with high correlation coefficients ( r 2 = 1.000). The proposed method was successfully applied to determine EAs in epoxy resin curing agents.

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.

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.

Cd-bound phytochelatins (Cd-PCs) have been synthesised by incubation of Phaeodactylum tricornutum cell cultures with Cd and purified by size-exclusion chromatography-UV-Vis. These complexes, which were identified in previous work, have now been used as model substances to develop and optimise ion-pair chromatography (IPC) coupled to inductively coupled plasma-mass spectrometry (ICP-MS) for analysis of Cd-PCs. Subsequent analysis of samples taken from Silene vulgaris plants cultivated under heavy metal stress conditions revealed Cd signals but no Cd-PC signals. By use of isotopically enriched ¹¹⁶Cd-PCs the sample preparation steps were verified to determine the stability of the analytes. We observed species transformation between Cd-PCs and other unidentified Cd complexes. Consequently, the kinetic and thermodynamic lability of Cd-PCs are decisive factors in their detection.

Fortunately, the intentional contamination of food or water supplies out of criminal or terroristic motivation is a rather rare event. However, in the face of asymmetric warfare and as the consequences of such an event would be severe, food defence as a necessary supplement to food safety is gaining increased attention. While some progress has been made in developing non-target detection devices, the contamination of food or water supplies using readily available rodenticides may still be revealed only by complex analytical techniques. The presented study therefore aimed to develop a quick and easy screening method for the detection of sixteen globally common rodenticides in foodstuffs. Robust operation with limited personnel and analytical resources were one benchmark to be met by the method, which uses a slightly modified QuEChERS (quick, easy, cheap, effective, rugged, safe) protocol for dispersive solid-phase extraction and subsequent ion-pair chromatography with diode-array and fluorescence detection. Quantification limits were as low as 5 µg/kg with satisfying bias (recovery) and repeatability rates of 77 to 117% and 1.8 to 17.1%, respectively. The developed method provides reliable and robust detection of these deadly poisons at toxic concentrations, which was demonstrated impressively in an improvised assault scenario.

Developing analytical techniques that align with green and sustainable chemistry principles is crucial in today's scientific landscape. This work introduces two innovative approaches for the simultaneous quantification of indacaterol (IND) and mometasone (MOM), a recently approved combination therapy for chronic obstructive pulmonary disease. These methods—rapid isocratic ion pair chromatography (IPC) and UV–visible spectrophotometry—demonstrate improved environmental sustainability, cost-effectiveness, and versatility compared to existing techniques. The optimized 4-min IPC method achieved excellent resolution (retention times 2.18 ± 0.1 min for IND and 3.95 ± 0.1 min for MOM), peak symmetry, and sensitivity. It utilizes a low-cost ion pair mobile phase of acetonitrile and acidified water containing 0.025% sodium dodecyl sulfate (50:50% v/v), making it suitable for laboratories with standard chromatographic instruments. The spectrophotometric approach offers two procedures: first derivative and ratio derivative methods. These serve as simplified, low-cost alternatives for resource-limited laboratories without access to advanced instruments. Both techniques feature simplified protocols that minimize extraction and fractionation steps. Comprehensive validation confirmed outstanding accuracy (98–102%) and precision (%2 <). Sustainability assessments using ComplexGAPI, AGREE, carbon footprint, BAGI, and RGB12 tools demonstrated enhanced environmental performance compared to existing methods. The IPC and spectrophotometry methods achieved greenness scores of 0.81 and 0.85, respectively, surpassing the 0.63–0.67 range of reported techniques. Additionally, they showed lower carbon footprints of 0.035 and 0.022 kg CO2 equivalent emissions per sample, compared to 0.079–0.092 kg for conventional procedures. The application of novel \"blueness\" and \"whiteness\" concepts using BAGI and RGB12 algorithms further confirmed superior sustainability, with scores of 87.5 & 90 for blueness and 88.1 & 89.8 for whiteness. Successfully applied to quantify IND and MOM in combined capsules, this work provides a model for eco-friendly pharmaceutical analysis that maintains high analytical reliability while improving sustainability metrics.

A multiple heart-cutting (mLC-LC) two-dimensional HPLC-UV achiral–chiral method for the direct analysis of branched-chain amino acids (BCAAs) in food supplements under environmentally friendly conditions was developed to cope with the very well-known limited chemoselectivity of chromatographic media for enantioselective analysis. Both achiral and chiral methods were developed in compliance with the main principles of green chromatography. The achiral analysis was performed isocratically with an optimized ion-pair reversed-phase (IP-RP) method based on a water/EtOH (95:5, v/v) mobile phase containing heptafluorobutyric acid (7 mM) as the IP agent. The achiral method was characterized by a very appreciable performance and was validated before the analysis of the real sample. High recovery values for all compounds (from 97% to 101%) were found in the interday evaluation. Additionally, low RSD% values in the long-term period were measured, in the range between 1.1% and 4.8%. Still, an LOQ value of 0.06 mg/mL was established for all compounds. The quantitative analysis of a commercial food supplement revealed that BCAAs were present in amounts very close to those declared by the producer. The enantioselective analysis was carried out through the application of the chiral ligand-exchange chromatography (CLEC) approach, using O-benzyl-(S)-serine ((S)-OBS, 0.5 mM) as the chiral selector and Cu(II) nitrate (0.25 mM) as the metal source in the eluent. Resolution and separation factor values up to 2.31 and 1.43, respectively, were obtained. The two chromatographic systems were connected through a six-port switching valve, and the developed two-dimensional mLC-LC method confirmed the absence of D-enantiomers of BCAAs in the food supplement, as reported in the manufacturer’s label.

Background and aims Besides general effect of organic residues on soil quality and plant crop, hormonal direct effect on plant growth by extracted humic acids of organic materials is interesting and profitable theme. In the present work, we studied on direct interaction between humic acid and root growth, depending on different origin of organic materials. Methods All extracted humic acids of four organic materials (sewage sludge, compost sewage sludge, municipal solid waste, compost municipal solid waste) were characterized chemically by elemental analyses, ion pair chromatography (ICP), size exclusion chromatography (HPSEC), solid-state nuclear magnetic resonance (13C-CPMAS-NMR) and quantification of IAA. Later, different morphological effects on maize (principal root growth, lateral root growth, root area, root mitosic site, root dry weight and H+-ATPase activity of plasma membrane) were analyzed. Results All humic acids samples promoted root growth and proton pump activity in maize vesicles, especially those composted samples, which contained more carboxylic groups and had a more hydrophobic character, produced preferentially morphological and biochemical effects.

Ultra high performance liquid chromatography hyphenated with quadrupole time-of-flight mass spectrometry was used to determine the products of the in vitro metabolism of phosphorothioate oligonucleotides. These compounds may be used during antisense therapy as synthetic fragments of genes. For this reason, both a sample preparation method and a qualification method were developed during this study. Liquid–liquid extraction, protein or oligonucleotide precipitation, and solid-phase extraction were tested and compared in order to select the method that yielded the highest recoveries. Ion pair chromatography was used for separation while mass spectrometry was applied for metabolite identification. The influence of the type of ion pair reagent used on the resolution and sensitivity was investigated. Results indicated that a mixture of 1,1,1,3,3,3-hexafluoro-2-propanol, N,N-dimethylbutylamine, and methanol was the best mobile phase for maximizing both of these parameters. The developed method was applied to investigate the compounds that form during the incubation of phosphorothioate oligonucleotides with human liver microsomes. Metabolites with short sequences were created after 8 hours, while oligonucleotides constructed from a large number of nucleotide units were obtained after 12 hours of incubation. Moreover, regardless of the length of the polynucleotide chain, metabolites were produced by the same mechanism: enzymatic cleavage at the 3′ end of the sequence.

Synthetic antisense phosphorothioate oligonucleotides (PS) have undergone rapid development as novel therapeutic agents. The increasing significance of this class of drugs requires significant investment in the development of quality control methods. The determination of the many degradation pathways of such complex molecules presents a significant challenge. However, an understanding of the potential impurities that may arise is necessary to continue to advance these powerful new therapeutics. In this study, four different antisense oligonucleotides representing several generations of oligonucleotide therapeutic agents were evaluated under various stress conditions (pH, thermal, and oxidative stress) using ion-pairing reversed-phase liquid chromatography tandem mass spectrometry (IP-RPLC-MS/MS) to provide in-depth characterization and identification of the degradation products. The oligonucleotide samples were stressed under different pH values at 45 and 90 °C. The main degradation products were observed to be losses of nucleotide moieties from the 3′- and 5′-terminus, depurination, formation of terminal phosphorothioates, and production of ribose, ribophosphorothioates (Rp), and phosphoribophosphorothioates (pRp). Moreover, the effects of different concentrations of hydrogen peroxide were studied resulting in primarily extensive desulfurization and subsequent oxidation of the phosphorothioate linkage to produce the corresponding phosphodiester. The reaction kinetics for the degradation of the oligonucleotides under the different stress conditions were studied and were found to follow pseudo-first-order kinetics. Differences in rates exist even for oligonucleotides of similar length but consisting of different sequences.