Reversed-phase Conditions Research Articles

Comparative analysis of feature annotation methods for SESI-HRMS in exhaled breath analysis

Secondary electrospray ionization coupled to high-resolution mass spectrometry (SESI-HRMS) is a powerful method for the analysis of exhaled breath in real time. However, feature annotation is challenging due to the flow-injection nature of the technique. To evaluate alternative methods for enhancing feature annotation, a study was conducted where the exhaled breath of sixteen subjects was condensed and analyzed using dynamic headspace vacuum in-trap extraction gas chromatography-mass spectrometry (DHS-V-ITEX-GC–MS) and liquid chromatography coupled to mass spectrometry (LC-MS) using polar and reverse-phase conditions along with a data-independent MS2-acquisition method based on multiple injections. The annotation results obtained from these methods were compared to those from SESI-HRMS. The use of these techniques on breath condensate is unprecedented. The GC–MS method primarily detected compounds of exogenous origin, particularly additives in oral hygiene products like menthol. On the other hand, LC-MS detected a vast number of features, especially with the utilized data-independent acquisition method. Chemical classes to these features were assigned in-silico. In positive ion mode, mostly amino acids and amines were detected, while the largest group in negative ion mode consisted of carboxylic acids. Approximately 25% and 5% of SESI features had a corresponding match with LC-MS and GC–MS. While both GC–MS and LC-MS methods partially overlapped with the SESI features, there was limited overlap of both in the mass-to-charge range from 150 to 200. In conclusion, both GC–MS and LC-MS analysis of breath condensate can serve as supplementary tools for annotating features obtained from SESI-MS. However, to increase confidence in the annotation results, combining these methods with additional on-line fragmentation techniques is recommended.

Chromatographic enantioresolution and stereochemical characterization of synthetic cannabinoid receptor agonists with Whelk-O®1 chiral stationary phases under mass spectrometry compatible reversed-phase conditions: A study case with seized samples

Chromatographic enantioresolution and stereochemical characterization of synthetic cannabinoid receptor agonists with Whelk-O®1 chiral stationary phases under mass spectrometry compatible reversed-phase conditions: A study case with seized samples

Chiral zwitterionic stationary phases based on Cinchona alkaloids and dipeptides: Application in chiral separation of dipeptides under reversed phase conditions

Chromatographic behavior of novel chiral stationary phases with bonded selectors based on Cinchona alkaloids modified with dipeptides was studied using dipeptides as probe molecules. Buffer-free and salt containing hydro-organic solutions were used as the mobile phases. The selectors exhibit pseudoenantiomeric behavior with respect to the L/D or LL/DD enantiomers and do not behave so with respect to the LD/DL enantiomers. The alkaloid part of the selectors is the driver of enantioselectivity, while the dipeptide substituent plays a modulating role. The quinidine-based selectors demonstrate stronger adsorption affinity and higher enantioselectivity as compared to the quinine-based selectors. The dipeptide analytes containing a glycyl fragment are weaker retained and their enantiomers are worse separated comparing to dipeptides with both units being larger amino acids. Moreover, a phenyl group in the structure of a dipeptide analyte facilitates enantioseparation. The effect of the mobile phase composition on retention depends on the hydrophobicity of an analyte. Hydrophobic dipeptides are better eluted by methanol-rich solvents, hydrophilic dipeptides are better eluted with water-rich solvents, and dipeptides with an intermediate hydrophobicity demonstrate a U-shaped or more complicated dependence of the retention factor on the percentage of methanol. Even a small buffer addition to the mobile phase decreases retention, but the ion-exchange mechanism was not confirmed. The effect of an electrolyte is rather due to the shielding of the charged groups of the selector reducing thereby electrostatic interaction between the selector and analyte. Efficiency of the novel columns is comparable to that of other brush-type chiral columns, the highest achieved number of the theoretical plates per 1 m varying between 30,000 and 40,000.

Graphene oxide-based stationary phase synthesis and separation of drug molecules for liquid chromatography (HPLC) analysis

A new graphene oxide-based liquid chromatography stationary phase was synthesized, characterized, and used for the first time in the separation of paracetamol and caffeine in pharmaceutical preparations under reversed phase conditions. For this purpose, graphene oxide was synthesized using the modified Hummers method, and Si-GO stationary phase was obtained by immobilizing graphene oxide on aminopropyl silica. The obtained graphene oxide, Si–GO and aminopropyl silica materials were characterized by XRD, SEM, TEM and FTIR analysis. Different mobile phases were used to separate drug molecules in order to determine the mechanism of interaction between drug molecules and the stationary phase. Once the appropriate solvent system was determined, optimum chromatographic parameters such as mobile phase composition (methanol percentage in water), flow rate and column temperature were determined. The linearity of the method was in the range of 0.989–0.997. The maximum relative standard deviation (% RSD) value was calculated as 0.54. Mean recoveries for paracetamol and caffeine were 98%–99%, respectively, and LOD and LOQ were 6 × 10−3 to 6.5 × 10−3 mg/L and 2 × 10−2 to 2.2 × 10−2 mg/L, respectively. The results showed that the new graphene oxide-based stationary phase could be used for the separation of paracetamol and caffeine in pharmaceutical preparations.

Chemo- and enantioselective analysis of high hydrophilic organophosphate pesticides by liquid chromatography under different elution modes

Acephate and malathion are prevalent chiral organophosphate insecticides, giving rise to more toxic chiral metabolites such as methamidophos and malaoxon, respectively. Enantioselective separation of chiral pesticides is crucial due to the differing pharmacological, environmental, and toxicological profiles of enantiomers. This study systematically evaluates an enantio‑ and chemoselective performance of eight polysaccharide-based chiral columns (Chiralcel OB-H, Chiralcel OD-H, Chiralcel OJ-R, Chiralpak AD-H, Chiralpak AS-H, Chiralpak IG-3, Chiralpak IK-3, and Lux-Amylose-2) and ten mobile phases across multimodal elution (normal-phase, polar organic, and reversed-phase conditions). Within the 80 combinations per analyte, the chiral screening highlights specific enantioselectivity trends concerning hydrophilic and hydrophobic compounds when utilizing amylose- or cellulose-based polymers under different conditions. Notably, a set of columns including IK-3, OJ-R, AS-H, and AD-H demonstrated superior coverage, achieving at least a 62 % success rate for enantioseparation. For the resolution between the pairs acephate/methamidophos and malathion/malaoxon, where no further optimizations were undertaken, the success rate for enantio‑ and chemoselectivity was 10 % and 30 %, respectively.

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.

Dimethyl carbonate as a green alternative to acetonitrile in reversed-phase liquid chromatography. Part II: Purification of a therapeutic peptide

Preparative liquid chromatography in reversed phase conditions (RPLC) is the most common approach adopted in the downstream processing for the purification of therapeutic peptides at industrial level. Due to the strict requirements on the quality imposed by the Regulatory Agencies, routinary methods based on the use of aqueous buffers and acetonitrile (ACN) as organic modifier are commonly used, where ACN is practically the only available choice for the purification of peptide derivatives. However, ACN is known to suffers of many shortcomings, such as drastic shortage in the market, high costs and, most importantly, it shows unwanted toxicity for human health and environment, which led it among the less environmentally friendly ones. For this reason, the selection of a suitable alternative becomes crucial for the sustainable downstream processing of peptides and biopharmaceuticals in general.In this paper, a promising green solvent, namely dimethyl carbonate (DMC) has been used for the separation of a peptide not only in linear conditions but also for its purification through non-linear overloaded chromatography. The performance of the process has been compared to that achievable with the common method where ACN is used as organic modifier and to that obtained with two additional solvents (namely ethanol and isopropanol), already used as greener alternatives to ACN.This proof-of-concept study showed that, thanks to its higher elution strength, DMC can be considered a green alternative to ACN, since it allows to reduce method duration while reaching good purities and recoveries. Indeed, at a target purity fixed to 98.5 %, DMC led to the best productivity with respect to all the other solvents tested, confirming its suitability as a sustainable alternative to ACN for the purification of complex biopharmaceutical products.

Direct determination of tyrosine and tryptophane enantiomers in pumpkin (Cucurbita moschata) by HPLC-UV/Vis: Effect of cooking treatment on enantiomers profile

An analytical method to determine the content of tryptophan (Trp) and tyrosine (Tyr) enantiomers in pumpkin (Cucurbita moschata) is developed and validated. The method consists of a sample preparation procedure with a direct liquid chromatographic separation of enantiomers on a Whelk-O2 chiral stationary phase. The chromatographic separation was performed under gradient reversed-phase conditions in which the effect of pH and organic modifier were studied. A multivariate design of experiments was applied in the optimization of the extraction procedure. After optimization the complete analytical process was validated. Great accuracy was achieved with recovery between 92–109% with a relative standard deviation of less than 10%. Limits of detection obtained ranged between 0.04 and 0.07 mg L-1. The resulting analytical method is applied with success to dose the content of Trp and Tyr and their enantiomers in several pumpkin cultivars. The impact of several traditional cooking methods on the content of these amino acids was also studied. The obtained data showed that studied pumpkin is a good source of free Trp and Tyr, mainly L-Trp. The results confirm the natural presence of D-Trp in this vegetable. Finally, the results demonstrate that the levels of all analytes are modified during traditional cooking treatments.

Enantiomeric Separation and Degradation of Benoxacor Enantiomers in Horticultural Soil by Normal-Phase and Reversed-Phase High Performance Liquid Chromatography.

The separation of benoxacor enantiomers on six commercial chiral columns was investigated by high-performance liquid chromatography (HPLC) under normal-phase and reversed-phase conditions. The mobile phases included hexane/ethanol, hexane/isopropanol, acetonitrile/water, and methanol/water. The effects of the chiral stationary phases (CSPs), temperature, and mobile phase composition and ratio on the separation of benoxacor enantiomers were examined. Under normal-phase conditions, the two benoxacor enantiomers were completely separated on Chiralpak AD, Chiralpak IC, Lux Cellulose-1, and Lux Cellulose-3 columns and partially separated on a Lux Cellulose-2 column. Under reversed-phase conditions, benoxacor enantiomers were completely separated on a Lux Cellulose-3 column and partially separated on Chiralpak IC and Lux Cellulose-1 columns. Normal-phase HPLC performed better than reversed-phase HPLC for the separation of benoxacor enantiomers. As the column temperature increased from 10 °C to 4 °C, the enthalpy (ΔH) and entropy (ΔS) results indicated that the resolution was strongly affected by the temperature and that the lowest temperature did not always produce the best resolution. An optimized separation method on the Lux Cellulose-3 column was used to investigate the stability of benoxacor enantiomers in solvents and the degradation of benoxacor enantiomers in three types of horticultural soil. Benoxacor enantiomers were stable, and degradation or racemization were not observed in methanol, ethanol, isopropanol, acetonitrile, hexane, or water (pH = 4.0, 7.0, and 9.0). In three horticultural soils, the degradation rate of S-benoxacor was faster than that of R-benoxacor, resulting in soil enrichment with R-benoxacor. The results of this study will help to improve the risk assessment of enantiomer levels of benoxacor in the environment.

Enantioseparation of chiral phytocannabinoids in medicinal cannabis

The evaluation of the chiral composition of phytocannabinoids in the cannabis plant is particularly important as the pharmacological effects of the (+) and (-) enantiomers of these compounds are completely different. Chromatographic attempts to assess the presence of the minor (+) enantiomers of the main phytocannabinoids, cannabidiolic acid (CBDA) and trans-Δ9-tetrahydrocannabinolic acid (trans-Δ9-THCA), were carried out on heated plant extracts for the determination of the corresponding decarboxylated species, cannabidiol (CBD) and trans-Δ9-tetrahydrocannabinol (trans-Δ9-THC), respectively. This process produces an altered phytocannabinoid composition with several new and unknown decomposition products. The present work reports for the first time the stereoselective synthesis of the pure (+) enantiomers of the main phytocannabinoids, trans-CBDA, trans-Δ9-THCA, trans-CBD and trans-Δ9-THC, and the development and optimization of an achiral-chiral liquid chromatography method coupled to UV and high-resolution mass spectrometry detection in reversed phase conditions (RP-HPLC-UV-HRMS) for the isolation of the single compounds and evaluation of their actual enantiomeric composition in plant. The isolation of the peaks with the achiral stationary phase ensured the absence of interferences that could potentially co-elute with the analytes of interest in the chiral analysis. The method applied to the Italian medicinal cannabis variety FM2 revealed no trace of the (+) enantiomers for all phytocannabinoids under investigation before and after decarboxylation, thus suggesting that the extraction procedure does not lead to an inversion of configuration.

Vacuum-assisted thermal bonding of β-cyclodextrin and its derivatives as chiral stationary phases for high-performance liquid chromatography

In this work, the vacuum-assisted thermal bonding method was proposed for the covalent coupling of β-cyclodextrin (β-CD) (CD-CSP), hexamethylene diisocyanate cross-linked β-CD (HDI-CSP) and 3, 5-dimethylphenyl isocyanate modified β-CD (DMPI-CSP) onto the isocyanate silane modified silica gel. Under vacuum conditions, the side reaction due to the water residue from the organic solvent, air, reaction vessels and silica gel could be avoided, and the optimal temperature and time of vacuum-assisted thermal bonding method were determined as 160°C and 3 h. These three CSPs were characterized by FT-IR, TGA, elemental analysis and the nitrogen adsorption-desorption isotherms. The surface coverage of CD-CSP and HDI-CSP on silica gel was determined as ∼0.2 μmol m−2, respectively. The chromatographic performances of these three CSPs were systematically evaluated by separating 7 flavanones, 9 triazoles and 6 chiral alcohols enantiomers under the reversed-phase condition. It was found that the chiral resolution ability of CD-CSP, HDI-CSP and DMPI-CSP was complementary to each other. Among them, CD-CSP could separate all 7 flavanones enantiomers with the resolution of 1.09–2.48. HDI-CSP had a good separation performance for triazoles enantiomers with one chiral center. DMPI-CSP showed excellent separation performance for chiral alcohol enantiomers, among which the resolution of trans-1, 3-diphenyl-2-propen-1-ol reached 12.01. Generally, the vacuum-assisted thermal bonding had been demonstrated as a direct and efficient method for the preparation of chiral stationary phases of β-CD and its derivatives.

Nano-liquid chromatography with monolithic stationary phase based on naphthyl monomer for proteomics analysis

Monolithic poly(2-vinylnaphthalene-co-divinylbenzene) columns were introduced, for the first time, and were evaluated as the separation media for nano-liquid chromatography (nano-LC). These columns were prepared by in-situ polymerization of 2-vinylnaphthalene (2-VNA) as the functional monomer and divinylbenzene (DVB) as the crosslinker in a fused silica capillary column of 50 µm i.d. Various porogenic solvents, including tetrahydrofuran (THF), dodecanol and toluene were used for morphology optimization. Final monolithic column (referred to as VNA column) was characterized by using scanning electron microscopy (SEM) and chromatographic analyses. Alkylbenzenes (ABs), and polyaromatic hydrocarbons (PAHs) were separated using the VNA column while the column offered excellent hydrophobic and π-π interactions under reversed-phase conditions. Theoretical plates number up to 41,200 plates/m in isocratic mode for ethylbenzene could be achieved. The potential of the final VNA column was demonstrated with a gradient elution in the separation of six intact proteins, including ribonuclease A (RNase A), cytochrome C (Cyt C), lysozyme (Lys), β-lactoglobulin (β-lac), myoglobin (My) and α-chymotrypsinogen (α-chym) in nano LC system. The column was then applied to the peptide analysis of trypsin digested cytochrome C, allowing a high peak capacity up to 1440 and the further proteomics analysis of COS-7 cell line was attempted applying the final monolithic column in nano-LC UV system.

A chiral porous organic polymer COP-1 used as stationary phase for HPLC enantioseparation under normal-phase and reversed-phase conditions.

A spherical chiral porous organic polymer (POPs) COP-1 is synthesized by the Friedel-Crafts alkylation reaction of Boc-3-(4-biphenyl)-L-alanine (BBLA) and 4,4'-bis(chloromethyl)-1,1'-biphenyl (BCMBP), which was used as a novel chiral stationary phase (CSPs) for mixed-mode high-performance liquid chromatography (HPLC) enantioseparation. The racemic compounds were resolved in normal-phase liquid chromatography (NPLC) using n-hexane/isopropanol as mobile phase and reversed-phase liquid chromatography (RPLC) using methanol/water as mobile phase. The COP-1-packed column exhibited excellent separation performance toward various racemic compounds including alcohols, amines, ketones, esters, epoxy compounds, organic acids, and amino acids in NPLC and RPLC modes. The effects of analyte mass and column temperature on the separation efficiency of racemic compounds were investigated. In addition, the chiral resolution ability of the COP-1-packed column not only can be complementary in RPLC/NPLC modes but also exhibit a good chiral recognition complementarity with Chiralpak AD-H column and chiral porous organic cage (POC) NC1-R column. The relative standard deviations (RSD) (n = 5) of the retention time, resolution value, and peak area by repeated separation of 1-(4-chiorophenyl)ethanol are all below 3.0%. The COP-1 column shows high column efficiency (e.g., 17,320 plates m-1 for 1-(4-chlorophenyl)ethanol on COP-1 column in NPLC), high enantioselectivity, and good reproducibility toward various racemates. This work demonstrates that chiral POPs microspheres are promising chiral materials for HPLC enantioseparation.

Simultaneous LC-ESI-MS/MS Quantification of Levosimendan and Its Metabolites for Therapeutic Drug Monitoring of Cardiac Surgery Patients.

Levosimendan is used in severe chronic cardiac insufficiency, also within the peri-operative setting. Real-life pharmacokinetic data in surgical patients is lacking, making therapeutic drug monitoring (TDM) of levosimendan, its pharmacologically active metabolite OR-1896, and its intermediate OR-1855 important. A simultaneous highly sensitive quantification of levosimendan and its metabolites in small-volume samples has not yet been described. Here, levosimendan (LLOQ 0.450 nM), OR-1896, and OR-1855 (LLOQ both 1.0 nM) were successfully quantified by LC-ESI-MS/MS after liquid-liquid extraction in 300 µL of blood. A short C8 column under reversed-phase conditions enabled simultaneous and fast quantification of levosimendan in the negative and the metabolites in the positive ionization mode in a single run within 2 min. Interestingly and unexpectedly, constitutional isomers of levosimendan metabolites with identical mass transitions and similar retention times were observed in surgical patients’ samples, which we identified as the metamizole metabolites 4-aminoantipyrine and 4-acetamidoantipyrine. A longer C8 column and a modified mobile phase enabled selective quantification of all analytes in a single run within 7 min. We developed, validated, and applied highly sensitive LC-ESI-MS/MS methods for simultaneous quantification of levosimendan and its metabolites, enabling efficient TDM of cardiac surgery patients even with additional metamizole administration.

Simultaneous enantioselective determination of two succinate- dehydrogenase-inhibitor fungicides in plant-origin foods by ultra-high performance liquid chromatography−tandem mass spectrometry

Fluindapyr and penthiopyrad are two new succinate-dehydrogenase-inhibitor fungicides both employed as racemic mixtures of enantiomers to control various fungal pathogens. In the present work, a robust and highly-sensitive method for simultaneous determination of fluindapyr and penthiopyrad enantiomers in plant-origin foods (cereals, fruits and vegetables) was developed using UPLC-MS/MS combined with a chiral stationary phase. Rapid baseline chiral separation of four stereoisomers of fluindapyr and penthiopyrad was obtained within 4.2 min on chiral MX(2)-RH column under reversed-phase conditions (with the eluent of acetonitrile/0.1% formic acid in water =70/30 (V:V) and column temperature maintained at 30 °C). The plant-origin samples were extracted quickly with acetonitrile and purified with multi-walled carbon nanotubes. Excellent linearity for the target analytes was observed in the concentration ranging from 1 to 250 µg/L with regression coefficient no less than 0.9967. The mean recoveries of fluindapyr and penthiopyrad enantiomers from six matrices were 77.1–107.2%, with all relative standard deviations values lower than 9.1%. The limit of quantification of four stereoisomers of two target chiral fungicides was 5 µg/kg. The analysis of real samples reveal that the developed method is suitable for the simultaneous chiral determination of fluindapyr and penthiopyrad residues in cereals, fruits and vegetables samples at enantiomeric level and can support their further investigation on enantioselective environmental behaviors and residue surveillance.

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

The enantioseparation of five fluorinated β-phenylalanine analogs together with the nonfluorinated α- and β-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.

Preparation of chiral stationary phase based on a [3+3] chiral polyimine macrocycle by thiol-ene click chemistry for enantioseparation in normal-phase and reversed-phase high performance liquid chromatography

Polyimine macrocycles are a new class of organic macrocycles with cyclic structures, well-defined molecular cavities, and multiple cooperative binding sites, which have recently aroused considerable research interest in molecular recognition and separation. Herein, we report the bonding of a [3+3] chiral polyimine macrocycle (H3L, C78H78N6O3) on thiol-functionalized silica gel using thiol-ene click chemistry to prepare a chiral stationary phase (CSP) for high performance liquid chromatography (HPLC). The fabricated column exhibited excellent chiral separation capability under both normal-phase and reversed-phase conditions. Fourteen and 10 racemates were well resolved on the column in normal-phase mode (using n-hexane/isopropanol as the mobile phase) and reversed-phase mode (using methanol/water as the mobile phase), respectively, including alcohols, esters, ethers, ketones, aldehydes, epoxides and organic acids. Moreover, the column also shows good selectivity toward positional isomers. Six positional isomers (dinitrobenzene, chloroaniline, bromoaniline, iodoaniline, nitrobrobenzene and nitrochlorobenzene) were well separated on the column. In addition, the effects of the injection mass and mobile phase composition on the separation were investigated. The column shows good reproducibility and stability after multiple injections with the relative standard deviation (RSD) (n = 5) of the retention time and resolution being < 0.96 % and 0.65 %, respectively. This study indicates that this type of chiral polyimine macrocycles is a promising chiral selector for HPLC enantioseparation and will push forward the applications of more novel chiral macrocycles for chiral chromatographic separation.

Chiral separation of dipeptides on Cinchona-based zwitterionic chiral stationary phases under buffer-free reversed-phase conditions.

Chiral zwitterion ion exchangers represent efficient chiral stationary phases for stereoselective resolution of various analytes including chiral acids, bases, and zwitterions. In this contribution, we have focused on utilization of chiral zwitterionic sorbents, denoted as ZWIX (+A) and ZWIX (-A). These are analogical chiral systems to commercially available columns, Chiralpak ZWIX (+) and Chiralpak ZWIX (-), which are usually operated with buffered mobile phases. In this contribution, we have studied the enantiorecognition power of the ZWIX (+A) and ZWIX (-A) columns on a series of dipeptides operated under buffer-free reversed-phase conditions. Retention characteristics of zwitterionic dipeptides are discussed using an electrostatically driven adsorption model, which provides a good fit with both monotonous and U-shaped curves.

Investigation of retention behavior of natural cannabinoids on differently substituted polysaccharide-based chiral stationary phases under reversed-phase liquid chromatographic conditions

The growing popularity of cannabis products and recent legalization of cannabis for recreational purposes have contributed to the increase of the demand for analytical methods able to give a detailed characterization of cannabis samples and derivatives. In this context, one of the aspects that is strongly emerging is about the hazardous potential of uncharacterised minor cannabinoids, including chiral ones, for which achiral potency testing methods currently employed do not give any information. For this reason, there is a growing interest towards the development of liquid chromatographic methods for the enantioseparation of cannabinoids. Much work is needed in this field where one of the major limitations is the lack of optically pure standards. This manuscript reports about the chromatographic behavior of five popular cannabinoids (including the cannabichromene racemate, CBC) on nine immobilised polysaccharide-based chiral stationary phases (CSPs) differently substituted, under reversed phase conditions. Results showed that chemo-selectivity of CSPs is not affected by changes in mobile phase composition, in the range of mobile phase investigated. In addition, the presence of electron withdrawing groups on the CSPs systematically leads to shorter retention times compared to when electron donating groups are present. An application of separation of cannabinoids from a real hemp extract on two of the chiral columns employed in this work revealed the presence of both CBC enantiomers in the sample.

Reversed-phase chromatography as an effective tool for the chiral separation of anionic and zwitterionic carboranes using polysaccharide-based chiral selectors

The aspect of the different spatial arrangement of anionic cobalt bis(dicarbollides) and dicarba-nido-undecaboranes remains grossly overlooked despite increased scientific interest. Regarding their growing potential, which does not limit only to medicinal chemistry, suitable enantioseparation methods are needed. The presented paper explores the possibilities of chiral separations of anionic cobalt bis(dicarbollide) and dicarba-7,8-nido-undecaborane derivatives on four polysaccharide-based columns under reversed-phase conditions. The chromatographic behavior of anionic derivatives was evaluated and compared with that of zwitterionic clusters. The isocratic procedure for HPLC method development was suggested. The main parameters for the optimization of separations were described. Successful chiral separations were critically compared to previously reported results in normal phase and polar-organic mode. Reversed-phase separations are superior in resolution, lower consumption of organic solvents, and flexibility during the method development. Moreover, the first chiral discrimination of some hydroxyalkyl derivatives of anionic cobalt bis(dicarbollides) is reported. The outcomes of this work may be used for method development in any area, where the chirality of these interesting molecules is of concern.