Search Results (155)

Chiral liquid chromatography (cLC) using chiral stationary phases (CSPs) has become a crucial technique for separating enantiomers. Understanding enantiomeric discrimination is essential for improving chromatographic conditions and elucidating chiral molecular recognition; the computational methods are extremely helpful for this. To assess the relevance of the association of these two approaches and to analyze the current trends, in this review, a systematic analysis of the scientific literature was performed, covering recently published works (from 2015 to January 2025) on enantioseparation by cLC using CSPs and computational studies. CSPs based on polysaccharides and Pirkle-type were the most described (accounting for 52% and 14% of the studies, respectively). Regarding the computational methods, molecular docking and molecular dynamics (MD) were the most reported (accounting for 50% and 25% of the studies, respectively). In the articles surveyed, a significant growth in research concerning both cLC enantioseparation and computational studies is evident, emphasizing the benefit of the synergy between these two approaches. Full article

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

A direct enantio- and chemo-selective high-performance liquid chromatographic method was developed for determining the enantiomeric impurity of the chiral active pharmaceutical ingredient silodosin. The simultaneous separation of enantiomers of silodosin and its main organic related substances listed in the Japanese Pharmacopoeia (JP) monograph for drug substance was achieved on Chiralpak AD-3 (250 mm × 4.6 mm, 3 μm) column under normal-phase isocratic conditions. The optimized conditions employed the mixture n-heptane-ethanol-diethylamine (70:30:0.1) (v/v/v) as a mobile phase and a temperature of 35 °C. The complete separation of the enantiomers of silodosin and its main impurities was obtained within 12 min. The chromatographic method has been validated according to the International Conference on Harmonization (ICH) guidelines and compared with the method reported in the JP monograph. The standard curve for silodosin exhibited linearity (R² > 0.999) within the concentration range of 1.13–2500 µg mL⁻¹. The Chiralpak AD-3 has demonstrated a remarkable level of efficiency, enabling the attainment of limits of quantitation for silodosin of 1.13 µg mL⁻¹ (equivalent to 0.057% of a sample solution of 2 mg mL⁻¹) and ranging from 0.48 µg mL⁻¹ to 1.94 µg mL⁻¹ for other impurities. Full article

Background/Objectives: Latanoprost is a leading active pharmaceutical ingredient belonging to the synthetic prostaglandin F2α analogs, widely used as a first-line treatment for open-angle glaucoma and increased intraocular pressure. This study addresses the critical need for an accurate and precise chromatographic method that is capable of simultaneously quantifying latanoprost and six latanoprost-related substances in antiglaucoma eye drops. This will be crucial for patient safety and treatment efficacy. This method enables the separation of latanoprost isomers, (15S)-latanoprost, latanoprost enantiomer, and 5,6-trans latanoprost from latanoprost signal. Furthermore, it is specific for the well-known latanoprost degradants—the major latanoprost acid and the minor 15-ketolatanoprost—as well as synthetic derivatives, such as triphenylphosphine oxide (TPPO) and propan-2-yl 5-(diphenylphosphoryl)pentanoate (IDPP). Using forced degradation studies using high temperatures, UV light, alkalis, acids, and oxidizing agents, the degradation profiles of the drugs were characterized and the method’s stability-indicating power was confirmed. Methods: Separation was achieved on a stationary combined system comprising chiral and cyano columns. Reverse-phase gradient elution and UV 210 nm detection were employed. The novel method was validated according to the European Medicines Agency International Council for Harmonisation Q2 Validation of analytical procedures—Scientific guideline. Results: The method was shown to be linear in the range of 40–60 µg/mL for latanoprost and 0.05–2.77 µg/mL for related substances, confirmed by a correlation coefficient of r = 0.999. Recoveries for latanoprost were obtained within the range of 98.0–102.0% for assays and 90.0–110.0% for impurities. The detection and quantification limits for latanoprost were 0.025 µg/mL and 0.35 µg/mL, respectively. Conclusions: The analytical procedure developed is adequately sensitive, precise, and accurate compared to existing methods. The method can be reliably used to control the critical quality attributes of low-dose latanoprost products, ensuring their required high pharmaceutical quality, which translates into improvements in patient care. This advancement holds significant implications for enhancing the therapeutic management of glaucoma, ensuring drug safety and efficacy. Full article

Novel peptides based on common amino acid building blocks may serve as possible drug candidates; however, their flexible structures may require stabilization via the incorporation of conformational constraints. The insertion of unusual amino acids is a feasible option that may provide improved pharmacokinetic and pharmacodynamic properties of such peptide-type drugs. The stereochemical purity of these kinds of building blocks must be verified by an efficient separation technique, such as high-performance liquid chromatography. Here, we present and discuss the results of the stereoselective separation mechanism of ß-methylated phenylalanine (ß-MePhe), tyrosine (ß-MeTyr), 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (ß-MeTic), and cyclohexylalanine (ß-MeCha) together with non-methylated Phe, Tyr, Tic, and Cha applying Cinchona alkaloid-based chiral stationary phases (CSPs). The studied zwitterionic CSPs acting as ion exchangers provided optimal performance in the polar ionic mode when methanol or a mixture of methanol and acetonitrile was utilized as the mobile phase together with organic acid and base additives. It was found that the basicity of small amines applied as mobile phase additives did not directly influence the chromatographic ion exchange concept. However, the size of the amines and their concentration led to a reduced retention time following the principles of ion exchange chromatography. On the basis of a systematic study of the effects of the eluent composition on the chromatographic behavior, important structure–retention and enantioselectivity relationships could be revealed. Through a temperature study, it has become evident that the composition of the eluent and the structure of analytes markedly affect the thermodynamic properties. Full article

This review systematically summarizes the novel preparation methods of cyclodextrin-based chromatographic stationary phases and their applications for chiral recognition in separation techniques such as capillary gas chromatography and high-performance liquid chromatography. Aiming at the current situation that enantiomers of chiral compounds present significant differences at the pharmacological, pharmacodynamic, and toxicological levels, the core value of chromatographic chiral separation technology in the field of drug discovery and development is emphasized. By analyzing the unique cavity structure and excellent stereoselective properties of cyclodextrins, the mechanism of their action as a chromatographic stationary phase was elaborated. Combined with the typical applications of different derivatized cyclodextrin stationary phases in drug analysis, environmental testing, and biological samples, the value and potential of cyclodextrin stationary phases in stereoisomer separation are systematically demonstrated. Full article

Several D-amino acid residue-containing peptides (DAACPs) with antimicrobial, cardio-excitatory, and neuronal activities have been identified in various species. The L-Asn-D-Trp-L-Phe-NH₂ (N(dW)F) tripeptide, derived from Aplysia kurodai, exhibits cardiac activity in invertebrates. The chirality of the tryptophan residue at the second position in N(dW)F influences its conformation and biological characteristics. We demonstrated the chiral separation of N(dW)F and its diastereomer NWF using (S)-3,3′-diphenyl-1,1′-binaphthyl-20-crown-6-ether columns (CR-I(+)). A reduction in the ratio of acetonitrile and methanol in the mobile phase allowed the complete separation of N(dW)F and its diastereomer, improving the separation factor (α) from 0.96 to 6.28. Molecular dynamics simulations revealed that the interaction of N(dW)F with CR-I(−) was more favorable than with CR-I(+). These findings indicate that the structure of the CR-I column stereoselectively recognizes peptides and facilitates the separation of naturally occurring D-amino acid residue-containing tripeptides. 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

Midazolam is a benzodiazepine that is utilized for the induction of anesthesia and the facilitation of procedural sedation. Despite the absence of stereogenic centers, the non-planar seven-membered ring devoid of reflection symmetry elements confers planar stereogenicity to the molecule. Due to the rapid conformational inversion of the Rp and Sp enantiomers, which occurs via a simple ring flip, high-performance liquid chromatography (HPLC) enantiomeric separation is restricted to sub-room temperature conditions. In this study, the energy barriers for the racemization of midazolam at five distinct temperatures and in acetonitrile/water mixtures were determined by monitoring the decay of the circular dichroism signal at a specific wavelength over time. The kinetic and thermodynamic data obtained were compared with those determined by dynamic enantioselective high-performance liquid chromatography using the Chiralpak IG-3 chiral stationary phase, which contains the amylose tris(3-chloro-5-methylphenylcarbamate) as the selector. The temperature-dependent dynamic HPLC of midazolam was carried out at the same temperatures and with the same aqueous mixtures used in parallel kinetic off-column experiments. To simulate dynamic chromatographic profiles, a lab-made computer program based on a stochastic model was utilized. The results indicated that the moderate influence of the stationary phase resulted in a slight increase in the activation barriers, which was more pronounced as the time spent in the column increased. This phenomenon was found to be mitigated when switching from a 250 mm × 4.6 mm, 3 µm, Chiralpak IG-3 column to a 50 mm × 4.6 mm, 1.6 µm, Chiralpak IG-U UHPLC column. The outcomes obtained under UHPLC conditions were found to be more closely aligned with those obtained through the ECD technique, with a discrepancy of only 0.1 kcal/mol or less, indicating a high degree of concordance between the two methods. Full article

The present study is the first report on the chemical and enantiomeric compositions of essential oils from the Ecuadorian species Gynoxys hallii Hieron., Gynoxys calyculisolvens Hieron., and Gynoxys azuayensis Cuatrec. All the volatile fractions presented a sesquiterpene-based chemical profile, typical of other volatile fractions from this genus. Both qualitative (GC-MS) and quantitative (GC-FID) chemical analyses were carried out on two stationary phases of different polarity (non-polar and polar). The main constituents of G. hallii essential oil on the two columns, respectively, were α-pinene (33.6–31.5%), (E)-β-caryophyllene (6.2–6.4%), germacrene D (35.7–38.3%), and bicyclogermacrene (3.8–4.0%). In G. calyculisolvens, the major compounds were α-pinene (11.2–11.0%), p-cymene (4.0–3.7%), α-copaene (3.6–3.7%), (E)-β-caryophyllene (8.1–8.3%), germacrene D (20.8–22.0%), and germacrene D-4-ol (8.4–8.6%). Finally, the main components of G. azuayensis were α-pinene (4.5–4.1%), germacrene D (14.1–12.4%), bicyclogermacrene (2.6–3.0%), tridecanal (6.4–6.2%), and spathulenol (7.8–7.1%). Furthermore, enantioselective analyses were conducted on the three volatile fractions, using two stationary phases based on β-cyclodextrins. As a result, twelve chiral components were investigated, detecting both enantiomerically pure compounds and scalemic mixtures with various enantiomeric excess. 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

A good chiral separation usually results in a trial-and-error process; however, through systematic studies, certain principles can be established to correlate structure with chiral selectivity. These principles can then be applied to other chiral separations, reducing the time of developing chiral selective analytical methods. Using the model compounds, the established principles can be applied to a wider range of compounds. In this study, mandelic acid and its substituted derivatives were selected as model compounds to establish transferable rules. The chiral selectivity of 13 compounds was measured on various permethylated cyclodextrin selectors. Comparing the chiral selectivity of permethylated cyclodextrins with different ring sizes (α, β, and γ) provides further insight into the role of inclusion in the chiral selectivity of the cyclodextrin-based stationary phases. Different derivatives of acidic and hydroxyl functions of mandelic acids were tested. Ring- and alkyl-substituted mandelic acid enantiomeric pairs were also tested. By using these compounds, the role of hydrogen donor–acceptor interactions and dipole–dipole interactions and inclusions in chiral recognition processes were investigated. The chiral selectivity values were measured and extrapolated to the same temperature, for the sake of the comparison. Several general tendencies were concluded, which can be used for chiral separation of other enantiomer pairs. Full article

Tea tree oil (TTO), acquired from Melaleuca alternifolia (Maiden & Betche) Cheel, Myrtaceae, is a widely utilized essential oil (EO) due to its bioactive properties. The identification and quantification of TTO ingredients is generally performed by GC-MS, which provides the most accurate results. However, in some instances, the cost and time of analysis may pose a challenge. Thin-layer chromatography (TLC) and high-performance thin-layer chromatography (HPTLC) offer a simpler, faster, cost-effective alternative capable of simultaneously analyzing and quantifying multiple samples. In addition, for more complex oils, two-dimensional (2D) or multigradient development (MGD) TLC provide better separation. Nevertheless, further development is sometimes necessary for the isolation of comigrating components. This study showcases a combined 2D-MGD TLC/HPTLC method for the successful separation of TTO components of interest. While human error, limited separation, and the partial evaporation of volatile components may still present a challenge during the process, considerable recovery of mono- and sesquiterpenes was achieved. This protocol also resulted in the successful isolation of target oxygenated monoterpenes (OMs) producing highly pure terpinen-4-ol (100%) and α-terpineol (≥94%), confirmed by GC-MS. The accurate enantiomeric distribution of these major OMs was verified by GC-FID through the use of a chiral cyclodextrin-based stationary phase. The observed positive enantiomer range (area percent) as well as (+)/(−) ratio for each terpinen-4-ol and α-terpineol were within acceptable ISO criteria. Full article

This study presents the first chemical and enantioselective analyses of essential oils (EOs) derived from the leaves of two endemic species, Gynoxys reinaldii Cuatrec. and Gynoxys pulchella (Kunth) Cass., from Loja, Ecuador. The distillation yields, by weight of dry plant material, were 0.04 ± 0.007% for G. reinaldii and 0.03 ± 0.002% for G. pulchella. For both plants, the chemical analyses were conducted by GC-MS (qualitative) and GC-FID (quantitative), on two stationary phases of different polarity (5% phenyl-methylpolysiloxane and polyethylene glycol). The major components of G. reinaldii EO included germacrene D (22.3–22.1%), α-pinene (14.2–14.1%), and (E)-β-caryophyllene (13.6–14.5%). Similarly, G. pulchella EO was characterized by germacrene D (9.5–12.9%), caryophyllene oxide (7.2–6.7%), and n-tricosane (4.9% in both columns). The enantioselective analyses were carried out with two columns, based on 2,3-diacetyl-6-tert-butyldimethylsilyl-β-cyclodextrin and 2,3-diethyl-6-tert-butyldimethylsilyl-β-cyclodextrin, detecting nine chiral terpenes and terpenoids. In G. reinaldii EO, (1S,5S)-(−)-α-pinene, (1S,5S)-(−)-β-pinene, (1S,5S)-(−)-sabinene, (R)-(−)-α-phellandrene, and (R)-(−)-β-phellandrene were enantiomerically pure, whereas cis-linalool oxide, linalool, terpinene-4-ol, and germacrene D were non-racemic mixtures of enantiomers. In G. pulchella, only (R)-(−)-α-phellandrene was enantiomerically pure. The detection of enantiomerically pure compounds may provide insights into the biosynthetic pathways and potential bioactivities of these EOs. Full article

Lathyrus odoratus L., commonly known as sweet pea, is a plant with a distinctive aroma that can develop in various habitats. An analysis of the aromatic profile of the species was conducted using the HS-SPME (solid-phase microextraction headspace) technique. This study aimed to explore the composition of and variation in the floral scent emissions of L. odorathus. The floral scents from fresh flowers were collected over different months and analyzed using gas chromatography coupled with mass spectrometry on apolar and polar stationary phase columns. In the apolar column, the majority compounds included linalool (19.27–5.79%), α-trans-bergamotene (29.4–14.21%), and phenyl ethyl alcohol (30.01–1.56%), while on the polar column, the predominant compounds included myrcene (13.25%), (E,E)-α-farnesene (26.33–8.16%), α-trans-bergamotene (42.09–24.82%), and others. This investigation was complemented by enantioselective analysis using a chiral phase based in cyclodextrins, which revealed the presence of (1R)-(+)-α-pinene, (S)-(−)-limonene, (R)-(+)-germacrene D, and (R)-(E)-nerolidol as enantiomerically pure components and linalool as a racemic mixture. Notably, the principal component analysis (PCA) and heatmap revealed variations among the chemical compounds collected at different harvest times. This demonstrates that temporal factors indeed impact chemical compound production. Furthermore, research on the aromatic properties of flowers provides a theoretical basis for studying and improving the components of their scent. Full article