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The retention behavior in supercritical fluid chromatography and its stability over time are still unsatisfactorily explained phenomena despite many important contributions in recent years, especially focusing on linear solvation energy relationship modeling. We studied polar stationary phases with predominant –OH functionalities, i.e., silica, hybrid silica, and diol columns, and their retention behavior over time. We correlated molecular descriptors of analytes with their retention using three organic modifiers of the CO2-based mobile phase. The differences in retention behavior caused by using additives, namely, 10 mmol/L NH3and 2% H2O in methanol, were described in correlation to analyte properties and compared with the CO2/methanol mobile phase. The structure of >100 molecules included in this study was optimized by semiempirical AM1 quantum mechanical calculations and subsequently described by 226 molecular descriptors including topological, constitutional, hybrid, electronic, and geometric descriptors. An artificial neural networks simulator with deep learning toolbox was trained on this extensive set of experimental data and subsequently used to determine key molecular descriptors affecting the retention by the highest extent. After comprehensive statistical analysis of the experimental data collected during one year of column use, the retention on different stationary phases was fundamentally described. The changes in the retention behavior during one year of column use were described and their explanation with a proposed interpretation of changes on the stationary phase surface was suggested. The effect of the regeneration procedure on the retention was also evaluated. This fundamental understanding of interactions responsible for retention in SFC can be used for the evidence-based selection of stationary phases suitable for the separation of particular analytes based on their specific physicochemical properties.

The effect of makeup solvent composition in ultrahigh-performance supercritical fluid chromatography-triple quadrupole mass spectrometry using electrospray ionization was studied using a set of 91 compounds, 3 stationary phases, and 2 organic modifiers of the mobile phase. The 24 tested makeup solvents included pure alcohols and methanol in combination with commonly used additives such as water, formic and acetic acid, ammonia, and ammonia salts with varying molarity. The behavioral trends for different makeup solvent additives were established in the first step. Subsequently, the correlations between physicochemical properties and the MS responses were calculated using the Pearson correlation test and matrix plots. The regression analysis was performed using five descriptors: molecular weight, pKa, log P, number of hydrogen donors/acceptors, and the MS responses obtained with methanol as the makeup solvent. The resulting regression equations had a high prediction rate calculated as R2-predicted coefficient, especially when 10 mmol/L ammonium in methanol was used as an organic modifier of the mobile phase in positive mode. The trueness of these equations was tested via the comparison between experimental and predicted responses expressed as R2. Values of R2> 0.8 were found for 88% of the proposed equations. Thus, the MS response could be measured using only one makeup solvent and the responses of other makeup solvents could be easily estimated. The suitability and applicability of determined regression equations was confirmed by the analysis of 13 blind probes, i.e., compounds not included in the original set of analytes. Moreover, the predicted and experimental responses followed the same increasing/decreasing trend enabling one to predict makeup solvent compositions leading to the highest sensitivity.

The organic polymer-based monolithic columns have been evaluated as the separation media for analysis of peptides using supercritical fluid chromatography–mass spectrometry (SFC–MS). We demonstrate for the first time the SFC–MS separation of a mixture of polypeptides carried out using poly(styrene-co-divinylbenzene) monolithic columns and carbon dioxide/methanol mobile phase. A gradient from 2 to 40% methanol modifier containing 0.1% TFA as an acidic additive was applied for the optimized elution and the separation was achieved in less than 3 min. Selected ion monitoring enabled detection of selected masses characteristic of three ionophoric pentadecapeptide antibiotics gramicidin A, B, and C and their two corresponding isoforms. Furthermore, their identity was confirmed through determination of their [M + 2H]2+, [M + 2Na]2+, and [M + H + Na]2+ions acquired by positive-ion electrospray ionization-mass spectrometry (ESI-MS).

A total of 20 derivatives (1–20) of the crinane-type alkaloid ambelline were synthesized. These semisynthetic derivatives were assessed for their potency to inhibit both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). To predict central nervous system (CNS) availability, logBB was calculated, and the data correlated well with those obtained from the parallel artificial membrane permeability assay (PAMPA). All compounds should be able to permeate the blood–brain barrier (BBB) according to the obtained results. A total of 7 aromatic derivatives (5, 6, 7, 9, 10, 12, and 16) with different substitution patterns showed inhibitory potency against human serum BuChE (IC50< 5 μM), highlighting the three top-ranked compounds as follows: 11-O-(1-naphthoyl)ambelline (16), 11-O-(2-methylbenzoyl)ambelline (6), and 11-O-(2-methoxybenzoyl)ambelline (9) with IC50values of 0.10 ± 0.01, 0.28 ± 0.02, and 0.43 ± 0.04 μM, respectively. Notably, derivatives 6, 7, 9, and 16displayed selective human BuChE (hBuChE) inhibition profiles with a selectivity index > 100. The in vitro results were supported by computational studies predicting plausible binding modes of the compounds in the active sites of hBuChE.

Complex analysis of plant extracts usually requires a combination of several analytical approaches. Therefore, in this study, we developed a holistic two-injection approach for plant extract analysis, which is carried out within one instrument without the need for any manual intervention during the analysis. Ultrahigh-performance supercritical fluid chromatography (UHPSFC) was employed for the analysis of 17 volatile terpenes on a porous graphitic carbon column within 7.5 min, followed by analysis on short diol column where flavonoids, phenolic acids, and terpenoic acids were analyzed within 15.5 min. A multimodal ionization source combining electrospray and atmospheric pressure chemical ionization (ESCi) was selected for mass spectrometry detection as a simultaneous ionization of both lipophilic and polar compounds was required. The quantitative aspects of the final UHPSFC-ESI/ESCi-MS/MS two-injection approach were determined, and it was applied to the analysis of Eucalyptus sp.extracts prepared by supercritical fluid extraction. Current methods reported in the literature typically require a labor-intensive combination of liquid and gas chromatography for the complex analysis of plant extracts. We present for the first time a new UHPSFC approach requiring only a single instrument that provides an alternative approach to the analysis of complex plant extracts.

Abstract The Horn of Africa supports a unique and rich diversity of squamate reptiles. Among them, the gecko genus Hemidactylusstands out as the most species-rich genus of the region. In this study, we assembled a genetic and morphological data set of 22 Hemidactylusspecies that form a clade termed the African radiation, which is part of the arid clade of the genus. We reconstructed their phylogenetic relationships using both concatenation-based approaches and species tree inference. Hemidactylus laevis, H. ophiolepoides, and H. somalicushave been sequenced and placed in a phylogenetic context for the first time in this study. Our results confirm the phylogenetic placement of these species within the African radiation of the genus. Early diversification within the clade, however, remains obscure. According to the analysis of concatenated data, H. laevisis sister to the rest of the clade, whereas the species tree analyses inferred the African radiation to be formed by two subclades, northern and southern, and H. laevisbelonging to the former. We integrate evidence from multiple sources including genetic differentiation at both mitochondrial and nuclear levels, morphological disparification, and coalescent-based species delimitation to support the existence of two new species of Hemidactylus. We provide a formal description of these two new species, one from northern Ethiopia, Eritrea, and westernmost Somaliland, and one from southern Ethiopia, northern Kenya, and easternmost South Sudan. Furthermore, we found that of the morphological traits examined, the numbers of supralabials and infralabials bear phylogenetic signal and we were able to tentatively infer the phylogenetic placement of species for which genetic data are still missing.

Etanercept is a recombinant Fc fusion protein widely used to treat rheumatic diseases. This protein is highly glycosylated and contains numerous O- and N-glycosylation sites. Since glycosylation is recognized as an important critical quality attribute (CQA) that can affect immunogenicity, solubility, and stability of Fc fusion proteins, it should be thoroughly characterized. In this work, hydrophilic interaction chromatography (HILIC) was combined with high-resolution mass spectrometry (HRMS) by using a quadrupole time-of flight mass spectrometer to assess glycosylation of etanercept at the middle-up level of analysis (fragments of ca. 25–30 kDa). In addition, a combination of different enzymatic digestion procedures (i.e., glycosidase, sialidase, and protease) was systematically employed to facilitate spectra deconvolution. With the developed procedure, the main post-translational modifications (PTMs) of etanercept were assessed, and a global overview of the subunit-specific distribution of the glycosylation pattern was obtained at a middle-up level of analysis.

Static and fatigue experiments were performed on friction stir (FS) welds in 2024-T3 aluminium alloys to investigate the influence of kissing bond (KB) flaws on the static strength and fatigue life of the FS welds. Different categories of FS welded plates were manufactured: a flaw-free plate and FS welded plates with different levels of KB size. The effects of the KB flaws on tensile and fatigue properties of AA2024-T3 specimens extracted from the plates were quantitatively evaluated with respect to the flaw-free weld. The various levels of KB flaw size were investigated with the aim of determining a flaw size that does not affect the fatigue life of the joined 6.35-mm-thick plates. The critical value for the KB geometry seems to be below 400 μm in depth based on the influence on the fatigue life for the investigated categories. The fatigue life of welds with KB flaws of 615 μm average depth was approximately seven times shorter than for flaw-free welds. The test results were supported by metallographic analyses and provide a baseline for definition of the flaw detection requirements related the sensitivity of non-destructive equipment.

A new ultra HPLC (UHPLC) method using both MS and fluorescence detection (FD) was developed for the determination of five fluoroquinolones in wastewaters. Systematic method development approach was compared with a conventional one. During the systematic approach, a possibility of automatic switching among four independent analytical columns of different chemistries has been used. Acidic as well as basic pH using ACN and methanol as organic modifiers was tested. The best separation of fluoroquinolones was obtained on phenyl analytical column at pH 10.5, which is a completely novel approach for separation of fluoroquinolones. Further, a new SPE procedure was developed for the sample preparation using basic pH as well. The sensitivity and selectivity of FD and MS detection were compared. FD at basic pH 10.5 demonstrated lower sensitivity than at acidic pH, which is conventionally performed. At basic pH, UHPLC‐MS/MS was found about two orders of magnitude more sensitive than FD. Both methods were validated and subsequently UHPLC‐FD method was used for the evaluation of stability of fluoroquinolones. UHPLC‐MS/MS method was used for the analysis of wastewater samples. Norfloxacin and ciprofloxacin were detected in samples of influent and effluent from wastewater treatment plant. Ofloxacin was detected only in influent from wastewater treatment plant.

Retention characteristics of ultra‐HPLC (UHPLC) hybrid stationary phases (bridged ethyl hybrid (BEH) and BEH Amide) were studied in hydrophilic interaction chromatography system in the group of polar basic pteridines (neopterin, biopterin, dihydroneopterin and dihydrobiopterin). The effect of mobile phase composition, buffer type, pH and concentration on retention of pteridines were examined in detail under UHPLC‐fluorescence detection and UHPLC‐MS conditions. The selectivity, retention properties and column performance were examined. BEH HILIC did not provide sufficient retention and selectivity for the separation of four pteridines under any tested conditions. BEH Amide provided strong retention for all pteridines especially at high pH values such as 9.8. However, at pH 9.8 the selectivity of separation for the pairs neopterin‐dihydroneopterin and biopterin‐dihydrobiopterin substantially decreased and resulted in very long analysis time. The best separation of four pteridine derivatives was obtained in the pH range 4.8–7.8 within a reasonable analysis time up to 8 min for UHPLC‐fluorescence detection using higher concentrations of ammonium acetate buffer and up to 4 min for UHPLC‐MS using lower concentrations of ammonium acetate.

Comparison of HPLC methods using conventional particle‐based and monolithic columns for determination of indomethacin and its two degradation products, viz. 5‐methoxy‐2‐methylindoleacetic acid and 4‐chlorobenzoic acid, was carried out. Ketoprofen was used as an internal standard for data evaluation throughout the study. Conventional separation was based on analytical column Zorbax SB‐Phenyl (75×4.6 mm; 3.5 μm particles) used with a mobile phase composition of acetonitrile and phosphoric acid 0.2% (50:50, v/v) and isocratic flow at 0.6 mL/min. Three different lengths of Chromolith columns RP‐18e (25×4.6 mm, 50×4.6 mm, and 100×3 mm) were tested with respect to the validation parameters peak asymmetry, resolution, height equivalent to a theoretical plate, repeatability, and after optimisation compared to values obtained using a conventional Zorbax column. The developed methods were used to determine all three compounds in a pharmaceutical formulation – Indobene gel. Chromatographic parameters were comparable to those of a conventional particle‐based column. The analysis time was shortened as expected (retention times were lowered by a factor of two). Moreover, the repeatability of peak areas and retention times obtained with a 50 mm monolithic column was greatly improved (RSD values were lower than 0.40%).

The continuous expansion of research in the field of stable carboranes and their wide potential in the drug design require carrying out fundamental studies regarding their chiral separations. Although supercritical fluid chromatography (SFC) is a viable technique for fast enantioseparations, no investigation concerning boron cluster compounds has been done yet. We aimed at the development of a straightforward method enabling chiral separations of racemic mixtures of anionic cluster carboranes and metallacarboranes that represent an analytical challenge. The fast gradient screening testing nine polysaccharide-based columns was used. The key parameters affecting the selectivity were the type of chiral selector, the type of alcohol, and the base in cosolvent. Moreover, the addition of acetonitrile or water to the cosolvent was identified as an effective tool for decreasing the analysis time while preserving the resolution. After the optimization, the chiral separations of 19 out of 20 selected compounds were achieved in less than 10 min. These results demonstrate the clear advantage of SFC over chiral separations using HPLC in terms of both analysis time and structural variety of successfully separated compounds.

The performance of the current bottom-up liquid chromatography hyphenated with mass spectrometry (LC-MS) analyses has undoubtedly been fueled by spectacular progress in mass spectrometry. It is thus not surprising that the MS instrument attracts the most attention during LC-MS method development, whereas optimizing conditions for peptide separation using reversed-phase liquid chromatography (RPLC) remains somewhat in its shadow. Consequently, the wisdom of the fundaments of chromatography is slowly vanishing from some laboratories. However, the full potential of advanced MS instruments cannot be achieved without highly efficient RPLC. This is impossible to attain without understanding fundamental processes in the chromatographic system and the properties of peptides important for their chromatographic behavior. We wrote this tutorial intending to give practitioners an overview of critical aspects of peptide separation using RPLC to facilitate setting the LC parameters so that they can leverage the full capabilities of their MS instruments. After briefly introducing the gradient separation of peptides, we discuss their properties that affect the quality of LC-MS chromatograms the most. Next, we address the in-column and extra-column broadening. The last section is devoted to key parameters of LC-MS methods. We also extracted trends in practice from recent bottom-up proteomics studies and correlated them with the current knowledge on peptide RPLC separation.

Hydrophilic interaction liquid chromatography (HILIC) method using internal standard for the determination and stability study of ascorbic acid was developed. HILIC method was very fast and simple using the following analytical conditions: ZIC HILIC (150×2.1 mm, 3.5 μm) chromatographic column and mobile phase composed of ACN and 50 mM ammonium acetate buffer pH 6.8 (78:22 v/v). Diode array detection was performed and chromatograms were processed at 268 nm, the maximum wavelength of absorbance of ascorbic acid. An extensive stability study of ascorbic acid as a function of various factors including temperature, stabilizing agents, oxygen presence and its concentration in solution was performed in order to gain information about the quantitative influence of individual stability factors. Low temperature and stabilizing agents (o‐phosphoric acid and oxalic acid) were found to be key factors enabling substantial enhancement of the stability of ascorbic acid.

Miniaturization embracing instrumentation, column particle size, and column dimensions is one of the major current trends in separation techniques. This leads to shortening of analysis time and great savings in solvent consumption. Ultra performance liquid chromatography (UPLC) is one of the new developments in liquid chromatography. An ultra‐high pressure system allows using of small particle‐packed columns with small diameter, which has a positive effect on both system efficiency and analysis time. An analytical method for determination of the active substance diclofenac, the degradation product 1‐(2,6‐dichlorphenyl)‐2‐indolinone, and the preservatives methylparaben and propylparaben was used for testing and comparing LC systems. Various octadecylsilica‐based analytical columns were examined. Acquity UPLC BEH C18 (2.1×50 mm, 1.7 μm) and (2.1×100 mm, 1.7 μm) were tested for UPLC. The following analytical columns were used in a test for HPLC: Purospher RP 18e (125×4.0 mm, 5 μm), Zorbax Eclipse XDB C18 (75×4.6 mm, 3.5 μm), Zorbax Eclipse SB C18 (50×4.6 mm, 1.8 μm), as was a monolithic column (Chromolith Performance RP‐18e (100×4.6 mm). Results of a System Suitability Test (SST) were calculated and compared for each chromatographic peak. System efficiency and analysis duration were compared with regard to solvent consumption and system maintenance

Cardioprotective activity of dexrazoxane (ICRF-187), the only clinically approved drug against anthracycline-induced cardiotoxicity, has traditionally been attributed to its iron-chelating metabolite. However, recent experimental evidence suggested that the inhibition and/or depletion of topoisomerase IIβ (TOP2B) by dexrazoxane could be cardioprotective. Hence, we evaluated a series of dexrazoxane analogues and found that their cardioprotective activity strongly correlated with their interaction with TOP2B in cardiomyocytes, but was independent of their iron chelation ability. Very tight structure–activity relationships were demonstrated on stereoisomeric forms of 4,4′-(butane-2,3-diyl)bis(piperazine-2,6-dione). In contrast to its rac-form 12, meso-derivative 11(ICRF-193) showed a favorable binding mode to topoisomerase II in silico, inhibited and depleted TOP2B in cardiomyocytes more efficiently than dexrazoxane, and showed the highest cardioprotective efficiency. Importantly, the observed ICRF-193 cardioprotection did not interfere with the antiproliferative activity of anthracycline. Hence, this study identifies ICRF-193 as the new lead compound in the development of efficient cardioprotective agents.

A known alkaloid (+)-chenabinol (1) and two new secobisbenzylisoquinoline alkaloids were isolated by standard chromatographic methods from the root bark of Berberis vulgarisL. The structures of the new alkaloids, named berkristine (2) and verfilline (3), were established by spectroscopic (including 2D NMR), and HRMS (ESI) methods. The alkaloids were tested for their inhibition activity of human cholinesterases and prolyl oligopeptidase. Compound 1 inhibited human butyrylcholinesterase with an IC50value of 44.8 ± 5.4 μM.