Search Results (42)

The separation of three proton pump inhibitors (omeprazole, lansoprazole, and rabeprazole) as exemplified molecules containing chiral sulfoxide groups was investigated in polar organic liquid chromatographic mode on seven different polysaccharide stationary phases (Chiralcel OD and OJ; Chiralpak AD, AS, and IA; Lux Cellulose-2 and -4). Different alcohols, such as methanol, ethanol, 1-propanol, 2-propanol, and their combinations, were used as eluents. After method optimization, semi-preparative enantioseparation was successfully applied for the three proton pump inhibitors to collect the individual enantiomers. A detailed investigation was conducted into elution order reversal, thermodynamic parameters, the effect of eluent mixtures, and the hysteresis of retention time and selectivity. Using Chiralpak AS, containing the amylose tris[(S)-α-methylbenzylcarbamate] chiral selector, the separation of the investigated enantiomers was achieved in all four neat eluents, with methanol providing the best results. In many cases, a reversal of the enantiomer elution order was observed. In addition to chiral-selector-dependent reversal, eluent-dependent reversal was also observed. Notably, even replacing methanol with ethanol altered the enantiomer elution order. Both enthalpy- and entropy-controlled enantioseparation were also observed in several cases; however, temperature-dependent elution order reversal was not. The hysteresis of retention and selectivity was further investigated on amylose-type columns in methanol–2-propanol and methanol–ethanol eluent mixtures. The phenomenon was observed on all amylose columns regardless of the eluent mixtures employed. Hystereticity ratios were calculated and used to compare the hysteresis behaviors of different systems. Multivariate statistical analysis revealed that Chiralpak AS exhibited the most distinct enantioselective behavior among the tested columns, likely due to the absence of a direct connection between the carbamate moiety and the aromatic substituent. The present study aided in understanding the mechanisms leading to enantiomer recognition, which is crucial for developing new chiral stationary phases and chiral HPLC method development in general. Full article

Since the 1990s, cannabis has experienced a gradual easing of access restrictions, accompanied by the expansion of its legalization and commercialization. This shift has led to the proliferation of cannabis-based products, available as cosmetics, food supplements, and pharmaceutical dosage forms. Consequently, there has been a growing demand for reliable and reproducible extraction techniques alongside precise analytical methods for detecting and quantifying cannabinoids, both of which are essential for ensuring consumer safety and product quality. Given the variability in extraction and quantification techniques across laboratories, significant attention has recently been directed toward method validation. Validated methods ensure precise cannabinoid measurement in cannabis-based products, supporting compliance with dosage guidelines and legal limits. Thus, this review highlights recent advancements in these areas, with a particular focus on High-Performance Liquid Chromatography (HPLC) coupled with Ultraviolet (UV) detection, as it is considered the gold standard for cannabinoid analysis included in cannabis monographs present in several pharmacopeias. The research focused on studies published between January 2022 and December 2024, sourced from PubMed, Scopus, and Web of Science, that employed an HPLC-UV analytical technique for the detection of phytocannabinoids. Additionally, the review examines cannabinoid extraction techniques and the validation methodologies used by the authors in the selected papers. Notably, ultrasound extraction has emerged as the most widely utilized technique across various matrices, with Deep Eutectic Solvents (DESs) offering a promising, efficient, and environmentally friendly extraction alternative. Analytical chromatographic separations continue to be predominantly conducted using C18 reversed-phase columns. Nevertheless, in recent years, researchers have explored various stationary phases, particularly to achieve the enantioseparation of cannabinoids. Full article

Broccoli, a highly valued Brassica vegetable, is renowned for its rich content of bioactive substances, including glucosinolates, phenolic compounds, vitamins, and essential minerals. Glucosinolates (GSLs), secondary plant metabolites, are particularly abundant in broccoli. The global consumption of broccoli has increased due to its high nutritional value. This review examines the essential bioactive compounds in broccoli and their biological properties. Numerous in vitro and in vivo studies have demonstrated that broccoli exhibits various biological activities, including antioxidant, anticancer, antimicrobial, anti-inflammatory, anti-obesity and antidiabetic effects. This review analyzes several aspects of the chemical and biological activity of GSLs and their hydrolysis products, isothiocyanates such as sulforaphane, as well as phenolic compounds. Particular emphasis is placed on sulforaphane’s chemical structure, the reactivity of its isothiocyanate fraction (-NCS), and given the different behavior of SFN enantiomers, a wide and detailed review of the chemical synthesis methods described, by microbial oxidation, or using a chiral ruthenium catalyst and more widely using chiral auxiliaries for synthesizing sulforaphane enantiomers. In addition, the methods of chiral resolution of racemates by HPLC are reviewed, explaining the different chiral fillers used for this resolution and a third section on resolution using the formation of diastereomeric complexes and subsequent separation on achiral columns. Additionally, this review highlights the presence of antimicrobial peptides in broccoli, which have shown potential applications in food preservation and as natural alternatives to synthetic antibiotics. The antimicrobial peptides (AMPs) derived from broccoli target bacterial membranes, enzymes, oxidative stress pathways and inflammatory mediators, contributing to their effectiveness against a wide range of pathogens and with potential therapeutic applications. Full article

The tetrahydroisoquinoline skeleton is a pharmacologically significant core structure containing chiral centers, making enantiomeric separation crucial due to the potentially distinct biological effects of each enantiomer. In this study, laudanosine (N-methyl-tetrahydropapaverine) and its three derivatives (6′-bromo-laudanosine, norlaudanosine, and N-propyl-norlaudanosine) were synthesized and used as model compounds to investigate chiral recognition mechanisms. Screening over twenty cyclodextrins (CyDs) as chiral selectors in capillary electrophoresis (CE), we found anionic CyDs to be the most effective, with sulfated-γ-CyD (S-γ-CyD) achieving a maximum R_s of 10.5 for laudanosine. Notably, octakis-(6-deoxy-6-(2-carboxyethyl)-thio)-γ-CyD (sugammadex, SGX), heptakis-(2,3-O-diacetyl-6-O-sulfo)-β-CD (HDAS), heptakis-(2,3-O-dimethyl-6-O-sulfo)-β-CD (HDMS), and octakis-(2,3-O-dimethyl-6-O-sulfo)-γ-CD (ODMS) provided excellent enantioseparation for all four analytes. Following HPLC screening on CyD-based and polysaccharide-based chiral stationary phases, semi-preparative HPLC methods using amylose and cellulose-based columns were optimized to isolate enantiomers. The purity of the isolated enantiomers was evaluated by HPLC, and their configurations were confirmed via circular dichroism spectroscopy. The isolated enantiomers allowed us to explore enantiomer migration order reversals in CE and enantiomer elution order reversal in HPLC. Further ¹H and 2D ROESY NMR experiments provided atomic-level insights into enantioselective complex formation, confirming enantiomer differentiation by SGX and elucidating the inclusion complex structure, where the ring C immersion into the CyD cavity is prevalent. Full article

Besides the well-known hallucinogenic ketamine, various novel ketamine derivatives are available on the illicit drug market, sold as designer drugs. Minor chemical changes to the parent compound aim to circumvent existing narcotic drug laws while mimicking the effects of the original substance. Ketamine and some of its derivatives possess a chiral centre and therefore exist as two enantiomers. While differences in the effects of S- and R-ketamine are well studied, this is not the case for ketamine derivatives. Therefore, the development and adaptation of suitable enantioseparation methods for those compounds is important to face the problems of the constantly changing drug market. In this study, different chiral separation methods for capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC) were tested on 11 ketamine derivatives. Some of them were enantioseparated for the first time due to their novelty. All compounds were at least partially separated on both instruments. HPLC separations were conducted using four different polysaccharide-based chiral stationary phases. Furthermore, an optical rotation detector coupled to the HPLC enabled the determination of the enantiomer elution order. In CE analysis, enantioseparation was achieved using 2% (w/v) acetyl-β-cyclodextrin or carboxymethyl- β-cyclodextrin in 10 mM di-sodium hydrogen phosphate as the background electrolyte in capillary electrophoresis. Full article

The enantioselective binding of three proton pump inhibitors (PPIs)—omeprazole, rabeprazole, and lansoprazole—to two key plasma proteins, α1-acid glycoprotein (AGP) and human serum albumin (HSA), was characterized. The interactions between PPI enantiomers and proteins were investigated using a multifaceted analytical approach, including high-performance liquid chromatography (HPLC), fluorescence and UV spectroscopy, as well as in silico molecular docking. HPLC analysis demonstrated that all three PPIs exhibited enantioseparation on an AGP-based chiral stationary phase, suggesting stereoselective binding to AGP, while only lansoprazole showed enantioselective binding on the HSA-based column. Quantitatively, the S-enantiomers of omeprazole and rabeprazole showed higher binding affinity to AGP, while the R-enantiomer of lansoprazole displayed greater affinity for AGP, with a reversal in the elution order observed between the two protein-based columns. Protein binding percentages, calculated via HPLC, were greater than 88% for each enantiomer across both transport proteins, with all enantiomers displaying higher affinity for AGP compared to HSA. Thermodynamic analysis indicated that on the HSA, the more common, enthalpy-controlled enantioseparation was found, while in contrast, on the AGP, entropy-controlled enantioseparation was observed. The study also identified limitations in using fluorescence titration due to the high native fluorescence of the compounds, whereas UV titration was effective for both proteins. The determined logK values were in the range of 4.47–4.83 for AGP and 4.02–4.66 for HSA. Molecular docking supported the experimental findings by revealing the atomic interactions driving the binding process, with the predicted enantiomer elution orders aligning with experimental data. The comprehensive use of these analytical methods provides detailed insights into the enantioselective binding properties of PPIs, contributing to the understanding of their pharmacokinetic differences and aiding in the development of more effective therapeutic strategies. Full article

The significance and necessity of separating enantiomers in food, pharmaceuticals, pesticides, and other samples remains constant and unrelenting. The successful chiral separation usually includes the application of a chiral auxiliary compound, known also as a chiral selector (CS), that forms complexes with enantiomers of different physicochemical properties, enabling efficient separation. While both native and substituted cyclodextrins (CDs) are commonly used as CSs, β-CD is undoubtedly the most popular one among them. This review includes recent advancements in the application of β-CD as a CS. While the theoretical background behind the enantioseparation is also part of this work, the main emphasis is put on the factors that affect the efficacy of this process such as temperature, pH, solvent, and the choice of other additives. Also, the different analytical methods: Nuclear Magnetic Resonance (NMR) spectroscopy, Capillary Electrophoresis (CE), fluorescence spectroscopy (FS), High-Performance Liquid Chromatography (HPLC), Isothermal Titration Calorimetry (ITC), and UV–vis spectroscopy, used for enantioseparation with the aid of β-CD as CS, are thoroughly compared. Also, since some of the chiral compounds have been studied in the context of their enantioseparation more than once, those works are compared and critically analyzed. In conclusion, while β-CD can be in most cases used as CS, the choice of the experimental conditions and method of analysis is crucial to achieve the success. Full article

Natural ectoine, (+)-(4S)-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid, is an extremely important small biomolecule belonging to the class of osmolytic/osmoprotective compounds. It stabilizes biomacromolecules such as DNA and proteins and protects them from denaturation by heat, dehydration, and UV radiation. The rapidly growing interest in this compound resulted in currently exclusive biotechnological production, while a chemical process along with enantioseparation as an alternative has not yet been established. An improved chemical synthesis of racemic ectoine starting from γ-butyrolactone in very good yield is described. Regioselective monoacetylation is achieved by the complexation of a copper(II)-ion with two molecules of 2,4-diamonobutyric acid in the key synthetic step. The racemic ectoine was synthesized with the aim of being successfully enantioseparated for the first time by high-performance liquid chromatography (HPLC) using a teicoplanin-based Chiral-T column in different solvent systems. The presence of (+)-ectoine was determined and quantified using an HPLC protocol on the Synergy Polar-RP column in fermentation broths inoculated with different strains of Streptomyces sp. bacteria isolated from the Adriatic Sea and grown on different NaCl concentrations. Full article

The subject of this study was to compare the enantioseparation of fourteen racemic esters that are liquid crystals. This study aimed to determine the difference in the enantioseparation of mixtures with protonated and fluorinated aliphatic chains and those with different orders of occurrence of benzene rings (benzoates and biphenylates). This research was carried out on two chiral polysaccharide columns: amylose tris(3-chloro-5-methylphenylcarbamate) (ReproSil Chiral MIG) and cellulose tris(3,5-dichlorophenylcarbamate) (ReproSil Chiral MIC). The columns were evaluated in HPLC separation. The analyses were performed in the normal- and reversed-phase systems. The mobile phase consisted of different solvent systems (acetonitrile/water and n-hexane/2-propanol) in different volume ratios to select optimal separation conditions. The main parameter evaluated in separating racemic mixtures was the resolution—Rs. All measurements were performed at 25 °C. The elution order was also determined. The highest value of resolution (over 11) and selectivity (over 3) was obtained for the ReproSil Chiral MIG column and the volume ratio of ACN:H₂O (95:5 v/v). Full article

Sulforaphane is a chiral phytochemical with chemopreventive properties. The presence of a stereogenic sulfur atom is responsible for the chirality of the natural isothiocyanate. The key role of sulfur chirality in biological activity is underscored by studies of the efficacy of individual enantiomers as chemoprotective agents. The predominant native (R) enantiomer is active, whereas the (S) antipode is inactive or has little or no biological activity. Here we provide an enantioselective high-performance liquid chromatography (HPLC) protocol for the direct and complete resolution of sulforaphane and its chiral natural homologs with different aliphatic chain lengths between the sulfinyl sulfur and isothiocyanate group, namely iberin, alyssin, and hesperin. The chromatographic separations were carried out on the immobilized-type CHIRALPAK IH-3 chiral stationary phase with amylose tris-[(S)-methylbenzylcarbamate] as a chiral selector. The effects of different mobile phases consisting of pure alcoholic solvents and hydroalcoholic mixtures on enantiomer retention and enantioselectivity were carefully investigated. Simple and environmentally friendly enantioselective conditions for the resolution of all chiral ITCs were found. In particular, pure ethanol and highly aqueous mobile phases gave excellent enantioseparations. The retention factors of the enantiomers were recorded as the water content in the aqueous-organic modifier (methanol, ethanol, or acetonitrile) mobile phases progressively varied. U-shaped retention maps were generated, indicating a dual and competitive hydrophilic interaction liquid chromatography (HILIC) and reversed-phase liquid chromatography retention mechanism on the CHIRALPAK IH-3 chiral stationary phase. Finally, experimental chiroptical studies performed in ethanol solution showed that the (R) enantiomers were eluted before the (S) counterpart under all eluent conditions investigated. Full article

Enantioseparation of nineteen liquid crystalline racemic mixtures obtained based on (R,S)-2-octanol was studied in reversed-phase mode on an amylose tris(3-chloro-5-methylphenylcarbamate) (ReproSil Chiral-MIG) and a cellulose tris(3,5-dichlorophenylcarbamate) (ReproSil Chiral-MIC). These polysaccharide-based chiral stationary phase (CSP) columns for High-Performance Liquid Chromatography (HPLC) were highly effective in recognizing isomers of minor structural differences. The mobile phase (MP), which consists of acetonitrile (ACN)/water (H₂O) at different volume ratios, was used. The mobile phases were pumped at a flow rate of 0.3, 0.5, or 1 mL·min⁻¹ with a column temperature of 25 °C, using a UV detector at 254 nm. The order of the elution was also determined. The chromatographic parameters, such as resolution (R_s), selectivity (α), and the number of theoretical plates, i.e., column efficiency (N), were determined. The polysaccharide-based CSP columns have unique advantages in separation technology, and this study has shown the potential usefulness of the CSP columns in separating liquid crystalline racemic mixtures belonging to the same homologous series. Full article

Mebendazole (MBZ) is a benzimidazole carbamate anthelmintic used worldwide for the treatment and prevention of parasitic disorders in animals and humans. A large number of in vivo and in vitro studies have demonstrated that MBZ also has anticancer activity in multiple types of cancers. After oral administration, the phenylketone moiety of MBZ is rapidly reduced to the hydroxyl group to form the chiral hydroxy metabolite (MBZ-OH). To the best of our knowledge, there is no information in the literature on the stereochemical course of transformation and the anthelmintic and antitumor activity of individual enantiomers of MBZ-OH. In the present study, we describe in detail the direct HPLC resolution of MBZ-OH on a 100 mm × 4.6 mm Chirapak IG-3 column packed with 3 μm silica particles containing amylose (3-chloro-5-methylphenylcarbamate) as a selector. At 25 °C and using pure methanol as the mobile phase, the enantioseparation and resolution factors were 2.38 and 6.13, respectively. These conditions were scaled up at a semi-preparative scale using a 250 mm × 10 mm Chiralpak IG column to isolate multi-milligram amounts of both enantiomeric forms of the chiral metabolite. The chiroptical properties of the collected enantiomers were determined and, through a theoretical study, were related to the more stable conformations of MBZ-OH. The first and second eluted enantiomers were dextrorotatory and levorotatory, respectively, in dimethylformamide solution. Finally, by recording the retention factors of the enantiomers as the water content in the water–acetonitrile mobile phases was progressively varied, U-shaped retention maps were generated, indicating a dual and competitive hydrophilic interaction liquid chromatography and reversed-phase liquid chromatography retention mechanism on the Chirapak IG-3 chiral stationary phase. Full article

In this paper, the preparation of three new polysaccharide-type chiral stationary phases (CSPs) based on levan carbamates (3,5-dimethylphenyl, 4-methylphenyl, and 1-naphthyl) is described. The enantioseparation of (±)-trans-β-lactam ureas 1a–h was investigated by high-performance liquid chromatography (HPLC) on six different chiral columns (Chiralpak AD-3, Chiralcel OD-3, Chirallica PST-7, Chirallica PST-8, Chirallica PST-9, and Chirallica PST-10) in the polar organic mode, using pure methanol (MeOH), ethanol (EtOH), and acetonitrile (ACN). Apart from the Chirallica PST-9 column (based on levan tris(1-naphthylcarbamate), the columns exhibited a satisfactory chiral recognition ability for the tested trans-β-lactam ureas 1a–h. Full article

A reversed-phase high-performance liquid chromatographic (HPLC) method was developed for the simultaneous determination of the potential impurities of dexketoprofen, including the distomer R-ketoprofen. After screening the separation capability of four polysaccharide columns (Lux Amylose-1, Lux Amylose-2, Lux Cellulose-1 and Lux Cellulose-2) in polar organic and in reversed-phase modes, appropriate enantioseparation was observed only on the Lux Amylose-2 column in an acidified acetonitrile/water mixture. A detailed investigation of the mobile phase composition and temperature for enantio- and chemoselectivity showed many unexpected observations. It was observed that both the resolution and the enantiomer elution order can be fine-tuned by varying the temperature and mobile phase composition. Moreover, hysteresis of the retention times and enantioselectivity was also observed in reversed-phase mode using methanol/water mixtures on amylose-type columns. This could indicate that the three-dimensional structure of the amylose column can change by transitioning from a polar organic to a reversed-phase mode, which affects the enantioseparation process. Temperature-dependent enantiomer elution order and rare enthalpic/entropic controlled enantioseparation in the operative temperature range were also observed in reversed-phase mode. To find the best methodological conditions for the determination of dexketoprofen impurities, a full factorial optimization design was performed. Using the optimized parameters (Lux Amylose-2 column with water/acetonitrile/acetic acid 50/50/0.1 (v/v/v) at a 1 mL/min flow rate at 20 °C), baseline separations were achieved between all compounds within 15 min. Our newly developed HPLC method was validated according to the current guidelines, and its application was tested on commercially available pharmaceutical formulations. According to the authors’ knowledge, this is the first study to report hysteretic behavior on polysaccharide columns in reversed-phase mode. Full article

A series of nine racemic trans-β-lactam ureas were analyzed for enantiomer separation by high-performance liquid chromatography (HPLC) and supercritical fluid chromatography (SFC). The separations were performed on three immobilized polysaccharide-based chiral analytical columns (CHIRAL ART Amylose-SA, CHIRAL ART Cellulose-SB and CHIRAL ART Cellulose-SC). In HPLC mode, a normal-phase consisting of n-hexane/2-PrOH (90/10, v/v), a polar organic mobile phase consisting of 100% MeOH or 100% EtOH, and a non-standard mobile phase consisting of 100% dimethyl carbonate (DMC) were investigated. In SFC mode, the mobile phases CO₂/alcohol (80/20, v/v) and CO₂/DMC/alcohol (MeOH or EtOH; 70/24/6, v/v/v or 60/32/8, v/v/v) were investigated. The best achieved enantioseparation of trans-β-lactam ureas was obtained with an Amylose-SA column. We have shown that the green solvent dimethyl carbonate (DMC) can be efficiently used as a mobile phase in HPLC mode as well as in SFC mode along with the addition of polar organic modifiers (MeOH or EtOH). Full article