Liquid Chromatographic Enantioseparation of Newly Synthesized Fluorinated Tryptophan Analogs Applying Macrocyclic Glycopeptides-Based Chiral Stationary Phases Utilizing Core-Shell Particles.

Due to the favorable features obtained through the incorporation of fluorine atom(s), fluorinated drugs are a group with emerging pharmaceutical importance. As their commercial availability is still very limited, to expand the range of possible candidates, new fluorinated tryptophan analogs were synthesized. Control of enantiopurity during the synthesis procedure requires that highly efficient enantioseparation methods be available. In this work, the enantioseparation of seven fluorinated tryptophans and tryptophan was studied and compared systematically to (i) develop analytical methods for enantioselective separations and (ii) explore the chromatographic features of the fluorotrytophans. For enantioresolution, macrocyclic glycopeptide-based selectors linked to core-shell particles were utilized, applying liquid chromatography-based methods. Application of the polar-ionic mode resulted in asymmetric and broadened peaks, while reversed-phase conditions, together with mobile-phase additives, resulted in baseline separation for all studied fluorinated tryptophans. The marked differences observed between the methanol and acetonitrile-containing eluent systems can be explained by the different solvation abilities of the bulk solvents of the applied mobile phases. Among the studied chiral selectors, teicoplanin and teicoplanin aglycone were found to work effectively. Under optimized conditions, baseline separations were achieved within 6 min. Ionic interactions were semi-quantitatively characterized and found to not influence enantiorecognition. Interestingly, fluorination of the analytes does not lead to marked changes in the chromatographic characteristics of the methanol-containing eluents, while larger differences were noticed when the polar but aprotic acetonitrile was applied. Experiments conducted on the influence of the separation temperature indicated that the separations are enthalpically driven, with only one exception. Enantiomeric elution order was found to be constant on both teicoplanin and teicoplanin aglycone-based chiral stationary phases (L < D) under all applied chromatographic conditions.

High-performance liquid chromatographic enantioseparation of azole analogs of monoterpene lactones and amides focusing on the separation characteristics of polysaccharide-based chiral stationary phases.

High-performance liquid chromatography-based enantioseparation of newly prepared azole analogs of monoterpene lactones and amides was studied. Effects of additives and mobile phase composition were evaluated both in normal and polar organic modes. Applying amylose tris-(3,5-dimethylphenylcarbamate) selector in normal and polar organic modes acid and base additives were found to affect the peak profiles, without significantly influencing the enantiorecognition ability of the studied selector. In most cases, differences observed in retention times and enantioselectivities were lower than 10 and 20 % under normal phase and polar organic conditions, respectively. Under normal phase conditions decreased retention was observed for all the studied analytes with increased eluent polarity. Interestingly, enantioselectivity was only slightly (<10 %) influenced by the variation in the n-hexane/2-propanol ratio between 80/20 and 20/80 v/v. In polar organic mode, five different neat solvents (acetonitrile, methanol, ethanol, 1-propanol, and 2-propanol) were tested, and the best results were obtained with acetonitrile and ethanol in the case of Lux Amylose-1 column with enantioresolutions most often above 2. Based on results obtained with amylose and cellulose-based columns the amylose tris-(3,5-dimethylphenylcarbamate) selector is found to offer a superior performance both in normal and polar organic modes. When evaluating the possible effects of the selector immobilization, no striking differences were found in the normal phase. Usually, enantioselectivities and resolutions were higher (10-20 %), while retention factors of the first peaks were lower (20-30 %), on the coated-type column. In contrast, in polar organic mode, the retention characteristics and enantiorecognition ability of the coated and immobilized selectors were heavily affected by the nature of the polar solvent. Special attention has been paid to the history-dependent behavior of polysaccharide-based selectors. A confidence interval-based evaluation is suggested to help comparison of the histereticity observed in different systems. Several examples are shown to confirm that the recently discovered hysteresis is a common characteristic of polysaccharide-based selectors.

Focusing on the 5F-MDMB-PICA, 4F-MDMB-BICA synthetic cannabinoids and their primary metabolites in analytical and pharmacological aspects.

Nowadays, more and more new synthetic cannabinoids (SCs) appearing on the illicit market present challenges to analytical, forensic, and toxicology experts. For a better understanding of the physiological effect of SCs, the key issue is studying their metabolomic and psychoactive properties. In this study, our validated targeted reversed phase UHPLC-MS/MS method was used for determination of urinary concentration of 5F-MDMB-PICA, 4F-MDMB-BICA, and their primary metabolites. The liquid-liquid extraction procedure was applied for the enrichment of SCs. The pharmacological characterization of investigated SCs were studied by radioligand competition binding and ligand stimulated [35S]GTPγS binding assays. For 5F-MDMB-PICA and 4F-MDMB-BICA, the median urinary concentrations were 0.076 and 0.312 ng/mL. For primary metabolites, the concentration range was 0.029-881.02* ng/mL for 5F-MDMB-PICA-COOH, and 0.396-4579* ng/mL for 4F-MDMB-BICA-COOH. In the polydrug aspect, the 22 urine samples were verified to be abused with 6 illicit drugs. The affinity of the metabolites to CB1R significantly decreased compared to the parent ligands. In the GTPγS functional assay, both 5F-MDMB-PICA and 4F-MDMB-BICA were acting as full agonists, while the metabolites were found as weak inverse agonists. Additionally, the G-protein stimulatory effects of the full agonist 5F-MDMB-PICA and 4F-MDMB-BICA were reduced by metabolites. These results strongly indicate the dose-dependent CB1R-mediated weak inverse agonist effects of the two butanoic acid metabolites. The obtained high concentration of main urinary metabolites of 5F-MDMB-PICA and 4F-MDMB-BICA confirmed the relevance of their routine analysis in forensic and toxicological practices. Based on in vitro binding assays, the metabolites presumably might cause a lower psychoactive effect than parent compounds.

Enantioseparation of a-substituted proline analogs with macrocyclic glycopeptide-based chiral stationary phases immobilized on superficially porous particles of silica applying liquid chromatography with ultraviolet and mass spectrometric detection.

In this study, the liquid chromatography-based direct enantioseparation of the stereoisomers of α-substituted proline analogs has been investigated utilizing chiral stationary phases with UV and/or mass spectrometric (MS) detection. Macrocyclic antibiotics, such as vancomycin, teicoplanin, modified teicoplanin, and teicoplanin aglycone, all covalently immobilized to 2.7 µm superficially porous silica particles have been applied as stationary phases. Mobile phases utilizing mixtures of methanol and acetonitrile with different additives (polar-ionic mode) were optimized during method development. Best separations were achieved with mobile phases of 100% MeOH containing either 20 mM acetic acid or 20 mM triethylammonium acetate. Special attention was given to the applicability of MS-compatible mobile phases. Acetic acid was found to be advantageous as a mobile phase additive for MS detection. Enantioselective chromatographic behaviors are interpreted based on the explored correlations between the analytes' structural features and those of the applied chiral stationary phases. For the thermodynamic characterization, separations were studied in the temperature range of 5-50 °C. Generally, retention and selectivity decreased with increasing temperature, and in most cases, enthalpy-driven enantiorecognition was observed, but entropic contributions also were present. Unexpectedly, unusual shapes for the van Deemter curves were registered in the kinetic evaluations. General trends could be observed in the enantiomeric elution orders: S < R on VancoShell and NicoShell, and opposite R < S on TeicoShell and TagShell columns.

Multi-round recycling of green waste for the production of iron nanoparticles: synthesis, characterization, and prospects in remediation.

Due to the widespread applications of metal nanoparticles (NPs), green synthesis strategies have recently advanced, e.g., methods that utilize extracts made from different plant wastes. A particularly innovative approach to reducing large amounts of available household/agricultural green wastes is their application in nanoparticle generation. Regarding this, the aim of our work was to examine the possibility of upgrading green nanoparticle syntheses from an innovative economic and environmental point of view, namely by investigating the multiple recyclabilities of green tea (GT), coffee arabica (CA), and Virginia creeper (Parthenocissus quinquefolia) (VC) waste residues for iron nanoparticle (FeNPs) synthesis. The plant extracts obtained by each extraction round were analyzed individually to determine the amount of main components anticipated to be involved in NPs synthesis. The synthesized FeNPs were characterized by X-ray powder diffraction and transmission electron microscopy. The activity of the generated FeNPs in degrading chlorinated volatile organic compounds (VOC) and thus their future applicability for remediation purposes were also assessed. We have found that VC and especially GT residues could be reutilized in multiple extraction rounds; however, only the first extract of CA was suitable for FeNPs' generation. All of the obtained FeNPs could degrade VOC with efficiencies GT1-Fe 91.0%, GT2-Fe 83.2%, GT3-Fe 68.5%; CA1-Fe 76.2%; VC1-Fe 88.2%, VC2-Fe 79.7%, respectively, where the number (as in GT3) marked the extraction round. These results indicate that the adequately selected green waste material can be reutilized in multiple rounds for nanoparticle synthesis, thus offering a clean, sustainable, straightforward alternative to chemical methods.

Macrocyclic glycopeptides- and derivatized cyclofructan-based chiral stationary phases for the enantioseparation of fluorinated ß-phenylalanine analogs.

The enantioseparation of five fluorinated ß-phenylalanine analogs together with the nonfluorinated α- and ß-phenylalanines has been investigated utilizing chiral stationary phases. The employed chiral selectors include macrocyclic antibiotics, such as vancomycin, teicoplanin, and teicoplanin aglycone, isopropyl carbamate functionalized cyclofructan-6, and Cinchona alkaloid-based tert.-butyl carbamate quinine, all covalently bonded to 2.7 µm superficially porous silica particles. The applied conditions included reversed-phase and polar-ionic modes where the vancomycin-, and the cyclofructan-6-based core-shell particles proved to offer suitable efficiency. Under reversed-phase conditions typical hydrophobic chromatographic behavior was observed, especially in the H2O/MeOH system. The improved selectivity with increasing MeOH content observed in polar ionic mode suggests that H-bonding may not play a major role in the chiral recognition. The stoichiometric displacement model was probed to gather information on the ionic interactions. The ion-exchange process was found to affect retention, but it has no essential contribution to chiral recognition. Without paying special attention to the optimization of the system volume of the UHPLC instrument plate heights varying in the range of 10-50 µm were obtained. In all cases, retention and selectivity decreased with increasing temperature, and enthalpy-driven enantiorecognition was observed. Elution sequences were determined in all cases.

Enantiomeric separation of newly synthesized amino, thio, and oxy derivatives of monoterpene lactones, amides, and ester applying polysaccharide-based chiral stationary phases in normal-phase mode.

New amino, thio, and oxy derivatives of monoterpene lactones, amides, and esters have been synthesized and their enantioselective separations were investigated on seven covalently immobilized polysaccharide-based chiral stationary phases. The effects of basic additives, different short-chain alcohols, and the influence of the temperature on the chromatographic behavior were studied. In addition, relationships between the structure of selector and selectand and the chromatographic parameters were explored to reveal mechanistic details of chiral recognition. Experiments were performed in the temperature range 10-50°C and thermodynamic parameters were calculated from plots of lnα versus 1/T. The separations were generally enthalpy-controlled, but entropy-driven separation was also observed. Special attention has been paid to the enantiomer elution order and several examples are shown how the structural characteristics of the selector, the nature, and the concentration of the polar modifier induce reversal of the enantiomer elution order in the case of the polysaccharide-based selectors.

Enantioselective high-performance liquid chromatographic separation of fluorinated ß- phenylalanine derivatives utilizing Cinchona alkaloid-based ion-exchanger chiral stationary phases: Enantioselective separation of fluorinated ß-phenylalanine derivatives.

The enantioselective separation of newly synthesized fluorine-substituted ß-phenylalanines has been performed utilizing Cinchona alkaloid-based ion-exchanger chiral stationary phases. Experiments were designed to study the effect of eluent composition, counterion content, and temperature on the chromatographic properties in a systematic manner. Mobile phase systems containing methanol or mixtures of methanol and acetonitrile together with acid and base additives ensured highly efficient enantioseparations. Zwitterionic phases [Chiralpak ZWIX (+) and ZWIX(-)] were found to provide superior performance compared to that by the anion-exchangers (Chiralpak QN-AX and QD-AX). A detailed thermodynamic characterization was also performed by employing van't Hoff analysis. Using typical liquid chromatographic experimental conditions, no marked effect of the flow rate could be observed on the calculated thermodynamic parameters. In contrast, a clear tendency has been revealed about the effect of the eluent composition on the thermodynamics for the zwitterionic phases.

Liquid Chromatographic Enantioseparations Utilizing Chiral Stationary Phases Based on Crown Ethers and Cyclofructans.

Natural compounds can exist in different forms, where molecules possessing chirality play an essential role in living organisms. Currently, one of the most important tasks of modern analytical chemistry is the enantioseparation of chiral compounds, in particular, the enantiomers of compounds having biological and/or pharmaceutical activity. Whether the task is to analyze environmental or food samples or to develop an assay for drug control, well-reproducible, highly sensitive, stereoselective, and robust methods are required. High-performance liquid chromatography best meets these conditions. Nevertheless, in many cases, gas chromatography, supercritical fluid chromatography, or capillary electrophoresis can also offer a suitable solution. Amino acids, proteins, cyclodextrins, derivatized polysaccharides, macrocyclic glycopeptides, and ion exchangers can serve as efficient selectors in liquid chromatography, and they are quite frequently applied and reviewed. Crown ethers and cyclofructans possessing similar structural characteristics and selectivity in the enantiodiscrimination of different amine compounds are discussed less frequently. This review collects information on enantioseparations achieved recently with the use of chiral stationary phases based on crown ethers or cyclofructans, focusing on liquid chromatographic applications.

Enantioseparation of ß2-amino acids by liquid chromatography using core-shell chiral stationary phases based on teicoplanin and teicoplanin aglycone.

Enantioseparation of nineteen ß2-amino acids has been performed by liquid chromatography on chiral stationary phases based on native teicoplanin and teicoplanin aglycone covalently bonded to 2.7 µm superficially porous silica particles. Separations were carried out in unbuffered (water/methanol), buffered [aqueous triethylammonium acetate (TEAA)/methanol] reversed-phase (RP) mode, and in polar-ionic (TEAA containing acetonitrile/methanol) mobile phases. Effects of pH in the RP mode, acid and salt additives, as well as counter-ion concentrations on chromatographic parameters have been studied. The structure of selectands (ß2-amino acids possessing aliphatic or aromatic side chains) and selectors (native teicoplanin or teicoplanin aglycone) was found to have a considerable influence on separation performance. Analysis of van Deemter plots and determination of thermodynamic parameters were performed to further explore details of the separation performance.

Enantioselective Liquid Chromatographic Separations Using Macrocyclic Glycopeptide-Based Chiral Selectors.

Numerous chemical compounds of high practical importance, such as drugs, fertilizers, and food additives are being commercialized as racemic mixtures, although in most cases only one of the isomers possesses the desirable properties. As our understanding of the biological actions of chiral compounds has improved, the investigation of the pharmacological and toxicological properties has become more and more important. Chirality has become a major issue in the pharmaceutical industry; therefore, there is a continuous demand to extend the available analytical methods for enantiomeric separations and enhance their efficiency. Direct liquid chromatography methods based on the application of chiral stationary phases have become a very sophisticated field of enantiomeric separations by now. Hundreds of chiral stationary phases have been commercialized so far. Among these, macrocyclic glycopeptide-based chiral selectors have proved to be an exceptionally useful class of chiral selectors for the separation of enantiomers of biological and pharmacological importance. This review focuses on direct liquid chromatography-based enantiomer separations, applying macrocyclic glycopeptide-based chiral selectors. Special attention is paid to the characterization of the physico-chemical properties of these macrocyclic glycopeptide antibiotics providing detailed information on their applications published recently.

Unexpected effects of mobile phase solvents and additives on retention and resolution of N-acyl-D,L-leucine applying Cinchonane-based chiral ion exchangers.

Chiral ion exchangers based on quinine (QN) and quinidine (QD), namely Chiralpak QN-AX and QD-AX as anionic and ZWIX(+) and ZWIX(-) as zwitterionic ion exchanger chiral stationary phases (CSPs) have been investigated with respect to their retention and chiral resolution characteristics. For the evaluation of the effects of the composition of the polar organic bulk solvents of the mobile phase (MP) and those of the organic acid and base additives acting as displacers necessary for a liquid chromatographic ion-exchange process, racemic N-(3,5-dinitrobenzoyl)leucine and other related analytes were applied. The main aim was to evaluate the impact of the MP variations on the observed, and thus the apparent enantioselectivity (αapp), and the retention factor. Significant differences were found using either polar protic methanol (MeOH) or polar non-protic acetonitrile (MeCN) solvents in combination with the acid and base additives as counter- and co-ions. It became clear, that the charged sites of both the chiral selectors of the CSPs and the analytes get specifically solvated, accompanied by the adsorption of all MP components on the CSP, thereby building a stagnant "stationary phase layer" with a composition different from the bulk MP. Via a systematic change of the MP composition, trends of resulting αapp and retention factors have been identified and discussed. In a detailed set of experiments, the effect of the concentration of the acid component in the MP containing MeOH or MeCN was specifically investigated, with the acid considered to be a displacer in anion-exchange type chromatographic systems. Surprisingly, all four chiral columns retained and resolved the tested N-acyl-Leu analytes with αapp values up to 21 within a retention factor window of 0.03 and 10 with pure MeOH as eluent. However, using pure MeCN as eluent, an almost infinite-long retention of the acidic analyte was noticed in all cases. We suggest that the rather different thickness of the solvation shells generated by MeOH or MeCN around the charged/chargeable sites of the chiral selector determines eventually the strength of the electrostatic selector-selectand interactions. As a control experiment we included the non-chiral N-acylglycine derivatives as analyte in all cases to support the interpretations with respect to the contribution of the enantioselective and non-enantioselective retention factor increments as a part of the observed αapp.

Cinchona-alkaloid-based zwitterionic chiral stationary phases as potential tools for high-performance liquid chromatographic enantioseparation of cationic compounds of pharmaceutical relevance.

Enantiomers of cationic compounds of pharmaceutical relevance, namely tetrahydro-ß-carboline and 1,2,3,4-tetrahydroisoquinoline analogs, were separated by high-performance liquid chromatography. Separations were performed on Cinchona-alkaloid-based zwitterionic ion exchanger type chiral stationary phases applied as cation exchangers using mixtures of methanol and acetonitrile or tetrahydrofuran as bulk solvent components containing triethylammonium acetate or ammonium acetate as organic salt additives. On the zwitterionic ZWIX(+) and ZWIX(-) columns investigated, retention and enantioseparation of the studied basic analytes were influenced by the nature and concentration of the organic components of the mobile phase. The effect of organic salt additives on the retention behavior of the studied analytes can be described by the stoichiometric displacement model related to the counterion concentration. Investigations on the structure-retention relationships were performed applying different mobile phase systems for the two types of cationic analytes. For the thermodynamic characterization, parameters such as changes in standard enthalpy (Δ(ΔH°)), entropy (Δ(ΔS°)), and free energy (Δ(ΔG°)) were calculated on the basis of van't Hoff plots derived from the ln α versus 1/T curves. In most cases, enthalpy-driven enantioseparations were observed, with a consistent dependence of the calculated thermodynamic parameters on the mobile phase composition. Elution sequences of the studied compounds were determined in all cases.

High-performance liquid chromatographic evaluation of strong cation exchanger-based chiral stationary phases focusing on stationary phase characteristics and mobile phase effects employing enantiomers of tetrahydro-ß-carboline and 1,2,3,4-tetrahydroisoquinoline analogs.

In this study, we present results obtained on the enantioseparation of some cationic compounds of pharmaceutical relevance, namely tetrahydro-ß-carboline and 1,2,3,4-tetrahydroisoquinoline analogs. In high-performance liquid chromatography, chiral stationary phases (CSPs) based on strong cation exchanger were employed using mixtures of methanol and acetonitrile or tetrahydrofuran as mobile phase systems with organic salt additives. Through the variation of the applied chromatographic conditions, the focus has been placed on the study of retention and enantioselectivity characteristics as well as elution order. Retention behavior of the studied analytes could be described by the stoichiometric displacement model related to the counter-ion effect of ammonium salts as mobile phase additives. For the thermodynamic characterization parameters, such as changes in standard enthalpy Δ(ΔH°), entropy Δ(ΔS°), and free energy Δ(ΔG°), were calculated on the basis of van't Hoff plots derived from the ln α vs. 1/T curves. In all cases, enthalpy-driven enantioseparations were observed with a slight, but consistent dependence of the calculated thermodynamic parameters on the eluent composition. Elution sequences of the studied compounds were determined in all cases. They were found to be opposite on the enantiomeric stationary phases and they were not affected by either the temperature or the eluent composition.

SZR-104, a Novel Kynurenic Acid Analogue with High Permeability through the Blood-Brain Barrier.

By being an antagonist of glutamate and other receptors, kynurenic acid serves as an endogenous neuroprotectant in several pathologies of the brain. Unfortunately, systemic administration of kynurenic acid is hindered by its low permeability through the blood-brain barrier. One possibility to overcome this problem is to use analogues with similar biological activity as kynurenic acid, but with an increased permeability through the blood-brain barrier. We synthesized six novel aminoalkylated amide derivatives of kynurenic acid, among which SZR-104 (N-(2-(dimethylamino)ethyl)-3-(morpholinomethyl)-4-hydroxyquinoline-2-carboxamide) proved to have the highest permeability through an in vitro blood-brain barrier model. In addition, permeability of SZR-104 was significantly higher than that of kynurenic acid, xanthurenic acid and 39B, a quinolone derivative/xanthurenic acid analogue. Since peripherally administered SZR-104 is able to inhibit epileptiform activity in the brain, we conclude that SZR-104 is a promising kynurenic acid analogue with good penetrability into the central nervous system.

Analytical Methodologies for the Characterization and Analysis of the Parent Compound and Phase I Metabolites of 4F-MDMB-BICA in Human Microsome, Urine, and Blood Samples.

4F-MDMB-BICA is one of the most dangerous new illicit synthetic cannabinoids (SCs) in 2020. Consumption of 4F-MDMB-BICA has been associated with a number of death cases and related serious adverse health effects in Hungary. Therefore, the use of reliable analytical methods to confirm the intake of 4F-MDMB-BICA is an important issue in forensic practice. Besides the detection of the parent compounds of SCs, the screening of their metabolites provides a reliable confirmation of their consumption, in particular, when the parent compound is under the limit of detection. To the best of our knowledge, this is the first report describing the identification of metabolites of 4F-MDMD-BICA after treatment with pooled human liver microsome (pHLM), and in human urine and blood samples using the combination of data obtained by comprehensive UHPLC-HRMS and semi-targeted UHPLC-HRMS/MS methods. Finally, our routine UHPLC-MS/MS method for screening urine and blood SCs was improved by adding the parent compound and selected main biomarkers of 4F-MDMD-BICA. From the pHLM assay of 4F-MDMD-BICA, 30 phase I metabolites were characterized and structural information thus obtained provided the basis of further identification of in vivo urine and blood metabolites. Overall, 20 urinary and 13 blood in vivo metabolites of 4F-MDMD-BICA have been identified by the investigation of five authentic urine and two blood samples. The ester hydrolysis metabolite was selected as a reliable primary biomarker in urine and blood. As secondary targets, urinary mono-hydroxylation metabolite and ester hydrolysis + dehydrogenation metabolite in blood were recommended due to their abundance and selectivity. Overall, the main phase I metabolites of 4F-MDMD-BICA were successfully characterized, and our routine analytical method with related sample preparation procedure provided a reliable analytical tool for screening both 4F-MDMD-BICA and its selected metabolites in urine and blood samples.

Polysaccharide-based chiral stationary phases as efficient tools for diastereo- and enantioseparation of natural and synthetic Cinchona alkaloid analogs.

In this study, we present results obtained on the diastereo- and enantioseparation of some basic natural and synthetic Cinchona alkaloid analogs by applying liquid chromatographic (LC) and subcritical fluid chromatographic (SFC) modalities on amylose and cellulose tris-(phenylcarbamate)-based stationary phases using n-hexane/alcohol/DEA or CO2/alcohol/DEA mobile phase systems. Seven chiral stationary phases in their immobilized form were employed to explore their stereoselectivity for a series of closely related group of analytes. The most important characteristics of LC and SFC systems were evaluated through the variation of the applied chromatographic conditions (e.g., the nature and content of the alcohol modifier, the concentration of additives, temperature). The columns Chiralpak IC and IG turned out to be the best in both LC and SFC modalities. Temperature-dependence study indicated enthalpy-controlled separation in most cases; however, separation controlled by entropy was also registered.

Heart-cutting two-dimensional liquid chromatography coupled to quadrupole-orbitrap high resolution mass spectrometry for determination of N,N-dimethyltryptamine in rat plasma and brain; Method development and application.

The orthogonal heart-cutting liquid chromatography (LC) modes coupled to high-resolution tandem mass spectrometry (HRMS/MS) provide a number of possibilities to enhance selectivity and sensitivity for the determination of targeted compounds in complex biological matricies. Here we report the development of a new fast 2D-LC-(HRMS/MS) method and its successful application for quantitative determination of the level of plasma and brain N,N-dimethyltriptamine (DMT) using α-methyltryptamine (AMT) as internal standard in an experimental model of cerebral ischemia/reperfusion using DMT administration. The 2D-LC separation was carried out by a combination of hydrophilic interaction liquid chromatography (HILIC) in the first dimension followed by second-dimensional reversed-phase (RP) chromatography within a total run time of 10 min. The enrichment of HILIC effluent of interest containing DMT was performed using a C18 trapping column. During method development several parameters of sample preparation procedures, chromatographic separation and mass spectrometric detection were optimised to achieve high DMT recovery (plasma: 90 %, brain: 88 %) and sensitivity (plasma: 0.108 ng/mL of LOD, brain: 0.212 ng/g of LOD) applying targeted analytical method with strict LC and HRMSMS confirmatory criteria. Concerning rat plasma sample, the concentration of DMT before hypoxia (49.3-114.3 ng/mL plasma) was generally higher than that after hypoxia (10.6-96.1 ng/mL plasma). After treatment, the concentration of DMT in brain was elevated up to the range of 2-6.1 ng/g. Overall, our analytical approach is suitable to detect and confirm the presence of DMT administered to experimental animals with therapeutic purpose in a reliable manner.

High-performance liquid chromatographic enantioseparation of isopulegol-based ß-amino lactone and ß-amino amide analogs on polysaccharide-based chiral stationary phases focusing on the change of the enantiomer elution order.

The enantioselective separation of newly prepared, pharmacologically significant isopulegol-based ß-amino lactones and ß-amino amides has been studied by carrying out high-performance liquid chromatography on diverse amylose and cellulose tris-(phenylcarbamate)-based chiral stationary phases (CSPs) in n-hexane/alcohol/diethylamine or n-heptane/alcohol/ diethylamine mobile phase systems. For the elucidation of mechanistic details of the chiral recognition, seven polysaccharide-based CSPs were employed under normal-phase conditions. The effect of the nature of selector backbone (amylose or cellulose) and the position of substituents of the tris-(phenylcarbamate) moiety was evaluated. Due to the complex structure and solvation state of polysaccharide-based selectors and the resulting enantioselective interaction sites, the chromatographic conditions (e.g., the nature and content of alcohol modifier) were found to exert a strong influence on the chiral recognition process, resulting in a particular elution order of the resolved enantiomers. Since no prediction can be made for the observed enantiomeric resolution, special attention has been paid to the identification of the elution sequences. The comparison between the effectiveness of covalently immobilized and coated polysaccharide phases allows the conclusion that, in several cases, the application of coated phases can be more advantageous. However, in general, the immobilized phases may be preferred due to their increased robustness. Thermodynamic parameters derived from the temperature-dependence of the selectivity revealed enthalpically-driven separations in most cases, but unusual temperature behavior was also observed.

Liquid chromatographic resolution of natural and racemic Cinchona alkaloid analogues using strong cation- and zwitterion ion-exchange type stationary phases. Qualitative evaluation of stationary phase characteristics and mobile phase effects on stereoselectivity and retention.

Liquid chromatographic (LC) and subcritical fluid chromatographic (SFC) resolution of the basic natural and synthetic Cinchona alkaloid analogues has been studied. Focus has been placed on the employment of four enantiomerically structured chiral strong cation-exchangers and four chiral diastereoisomeric Cinchona alkaloid and cyclohexyl aminosulfonic acid-based zwitterionic ion-exchangers. Except for the novel, recently synthesized racemic quinine the other investigated pairs of basic analytes are diastereomeric, but often called "pseudoenantiomeric" compounds of quinine and quinidine, cinchonidine and cinchonine, 9­epi­quinine and 9­epi­quinidine. As expected, the elution order of the resolved racemic quinine was reversed for all the eight investigated enantiomeric and (pseudo)enantiomeric pairs of chiral stationary phases, whereas this was not necessarily the case for the diastereomeric pairs of the Cinchona alkaloid related analytes. Varying the type and composition of the protic (methanol) and non-protic (acetonitrile) but polar bulk solvents in combination with organic salt additives in the mobile phase the overall retention and stereoselectivity characteristics could be triggered, leading to well performing LC and SFC systems. Thus the retention behavior of the basic analytes on both the chiral cation-exchangers and the diastereomeric zwitterionic ion-exchangers, used as cation-exchangers, could be described by the stoichiometric displacement model related to the counter-ion effect of the mobile phase additives. In addition, it became obvious that the non-protic acetonitrile compared to methanol as bulk solvent lead to a significant increase in retention, which can be associated with an increased electrostatic interaction of the charged sites due to a smaller solvation shell of the solvated cationic and anionic species. Based on the chromatographic results of the systematically selected chiral analytes and stationary phases attempts were undertaken to interpret qualitatively the observed stereoselectivity phenomena.