Chiral Method Research Articles

Chiral micellar electrokinetic chromatographic separation for determination of L- and D-primary amines released from murine islets of Langerhans.

D-amino acids have been located in various tissues including the endocrine portion of the pancreas, the islets of Langerhans. D-Serine (D-Ser), is of particular interest since it is an agonist for the ionotropic N-methyl-D-aspartate receptors. To examine the potential release of D-Ser and other D-amino acids from islets, a chiral micellar electrokinetic chromatography method was developed by derivatizing primary amines with 2,3-naphthalenedicarboxaldehyde and to achieve resolution of the enantiomers, two surfactants were used in the separation, sodium dodecyl sulfate and sodium deoxycholate. With the optimized conditions, 7 of 13 enantiomeric pairs that were tested had greater than baseline resolution, while the resolution of numerous other L-amino acids and small molecules were maintained. For the 17 compounds that were fully resolved, limits of detection were less than 10 nM. The resulting optimized separation method produced high efficiency peaks, with an average of 300,000 theoretical plates per peak and a peak capacity of 120. The method was used to examine the release of small molecules from groups of 50 murine islets of Langerhans. A peak was detected from islets incubated with 20 mM glucose that co-migrated with a D-Ser standard, although its level was below the quantifiable limit.

Separation and detection of cyproconazole enantiomers and its stereospecific recognition with chiral stationary phase by high-performance liquid chromatography.

Cyproconazole, a chiral triazole fungicide, has been diffusely used and analyzed. The development of an effective analytical method for cyproconazole enantiomers can support their residual monitoring and risk assessment. In the present study, the absolute configuration of the cyproconazole enantiomers was confirmed by electronic circular dichroism and time-dependent density functional theory. The enantioseparation parameters were optimized by the response surface methodology using the Box-Behnken design on Lux Cellulose-2. The elution order of (2S,3R)-(+)-, (2S,3S)-(+)-, (2R,3S)-(-)-, and (2R,3R)-(-)-cyproconazole was simulated with molecular docking. The enantiomers were completely separated primarily via halogen bond and hydrogen bond interactions with the chiral stationary phases. The mean recoveries of the cyproconazole enantiomers in the four matrices were 71.8-102.0% with intraday relative standard deviations (RSDs) of 0.3-11.9% and interday RSDs of 0.9-10.6%. The results showed the chiral recognition mechanism clearly and confirmed that the method was accurate and convenient for the simultaneous detection of cyproconazole enantiomers in environmental and food matrices.

Atropisomeric Separation of PCB-95 by HPLC

2,2’,3,5’,6-Pentachlorobiphenyl (PCB-95) is an environmentally significant chiral PCB, of which enantioselective toxicity, biodegradation and chiral stability studies have been limited to date, as no commercially available enantiomers exist for PCB-95 and due to the lack of an efficient preparatory chiral separation method. A selective, sensitive, and rapid high-performance liquid chromatography with UV detection (HPLC-UV) method has been developed and validated for the chromatographic separation and quantitation of PCB-95 enantiomers. In this study, we resolved enantiomers of PCB-95 using a cellulose tris (4-methylbenzoate) Chiralcel OJ- H column. After evaluating mobile phase compositions and temperatures, optimum separation and detection were obtained with isocratic 100% n-hexane as the mobile phase, a column temperature of 20°C, a flow rate of 1 mL/min, and a detection wavelength of 280 nm. The total run time was 8 minutes. Enantiomer purity was confirmed using enantioselective gas capillary chromatography-electron capture detection. The developed method was validated as per International Conference on Harmonization (ICH) guidelines with respect to limit of detection, limit of quantification, precision, linearity, robustness and ruggedness.

Applications of Chiral Supercritical Fluid Chromatography.

Nowadays polysaccharide chiral stationary phases are very popular and this originates for many reasons: (1) their wide applications window, (2) numerous different chemistry availability either in coated or in immobilized versions, and (3) large loading capability useful for preparative scale. Indeed chiral separations remain a hot topic (particularly in the pharmaceutical market) and in this field supercritical fluid chromatography is emerging rapidly. However, its use is more complex than high-performance liquid chromatography. The presented example illustrates analytical scale chiral separation method development and the effect of each operating parameter, i.e., flow rate, outlet pressure, and temperature variations on a chiral separation.

Enantiospecific UPC2 SFC-MS method for separation and quantification of R & S-Eliglustat tartrate in presence of its stereo isomers and degradation impurities

Abstract Eliglustat tartrate is an oral inhibitor of glucosylceramide synthase used in the treatment of Gaucher’s disease. This drug is administered as enantio-specific trans-(R,R) isomer and there is a possibility to have three more potential stereo isomers due to two chiral centers, owing to the toxicological difference between stereoisomers chiral method with high sensitivity is required to detect low level contaminations. A method for enantiomeric separation and quantification of trans-(R,R) Eliglustat tartrate in presence of other isomers trans-(S,S) Eliglustat, cis-(R,S) Eliglustat, cis-(S,R) Eliglustat was developed and validated using ultra-high performance supercritical fluid chromatography with tandem mass spectrometry (UPC2 SFC-MS) which is termed as a green chromatographic technique. Chiral separation was developed using Chiralcel OX-3 (4.6 * 150 mm) 3 µm column which has 4 chloro, 3 methyl phenyl carbamate as chiral selector, with a flow rate of 3 g/min using CO2 and 0.5% diethyl amine in ethanol: methanol (1:1) as a co-solvent. The effect on the enantioseparation by varying chiral additives, gradient conditions and content of organic modifier were investigated. The method has good linearity with concentration ranges from 0.25 to 7.5 µg mL−1. The detection limit was 0.08 µg mL−1 and quantification limit was 0.25 µg mL−1. Forced degradation studies were performed and all the degradation impurities were separated from product peak, the molecular weight of impurities were identified by using UPC2-Mass spectrometer. The method was validated in line to ICH guidelines in terms of linearity, accuracy, precision, LOQ, LOD and robustness.

Copper-dependent hydrolysis of trichloronate by turkey serum studied with use of new analytical procedure based on application of chiral chromatography and UV/Vis spectrophotometry

Trichloronate is a racemic organophosphate, which has been used for the manufacture of insecticides. This compound induces delayed neuropathy in hen and humans. This study shows the Cu2+-dependent hydrolysis of trichloronate by turkey serum using UV/Vis spectrophotometry and chiral chromatography. The CHIRALCEL OD column and mobile phase of heptane allowed a resolution of 1.15 of its two enantiomers, while the liquid-liquid extraction showed a recovery of 95–98%. The optimum linear response was of 50 to 800 μM with a detection and quantification limit of 0.6 and 2 μM for (+)-trichloronate, and 0.7 and 2.3 μM for (−)-trichloronate. The levels of Cu2+-dependent hydrolysis (μM remaining concentration) quantified for 60 min at 37 °C and pH 7.4 were statistically higher (p ˂ 0.05) for (−)-trichloronate (65%) than (+)-trichloronate (32%). This stereoselective hydrolysis was confirmed by UV/Vis spectrophotometry using 2,4,5‑trichlorophenol as standard, each of the enantiomers (93–95% purity) collected by HPLC, as well as aminoantipyrine and ferricyanide reagents to yield a colored product. This method exhibited an optimal linearity (r > 0.99) and a higher Cu2+-dependent hydrolysis (p < 0.05) to (−)-trichloronate (47%) than its corresponding (+)-form (31%). This results shows the Cu2+-dependent stereoselective hydrolysis of a racemic OP in its thio form (P = S) by an A-esterase of the turkey serum through the development of a colorimetric method and optimization of an existing chiral chromatographic method.

Stereoselectivity in a nitroso-ene cyclization: Formal synthesis of rac-manzacidins A and C

Nitroso-ene (NE) reactions have been adapted in the construction of nitrogen-containing motifs in many biologically interesting compounds. The transient and highly reactive “nitroso” species remains a challenge for designing stereocontrolled syntheses. Although chiral auxiliary-based method has been developed to achieve high diastereoselectivity, the use of the inherent chirality of the nitroso compound for stereochemical control remains underdeveloped. We chose the formation of a γ-lactam via a NE cyclization as a basis to study the applicable principles for future asymmetric syntheses. The selected examples presented herein reveal that the intramolecular hydrogen bond would provide conformational restraint to facilitate excellent facial selectivity in the NE reaction via a chair-like transition state. The sterically bulky amino group also provides excellent stereochemical control possibly through steric repulsion. The following transformations led to a key intermediate (Ohfune's lactone) in the preparation of manzacidins A and C and thus constitutes a formal synthesis.

Chiral separation of intact amino acids by capillary electrophoresis-mass spectrometry employing a partial filling technique with a crown ether carboxylic acid

An enantiomeric separation method for underivatized free amino acids (AAs) using a partial filling technique with CE-MS was developed for the determination of D-AAs in vinegars. A typical chiral separation method was performed with different concentrations of (18-crown-6)-2,3,11,12-tetracarboxylic acid (18C6H4) dissolved in water or formic acid as the background electrolyte. Seventeen AAs, excluding proline and asparagine, were separated, showing chiral resolution values (Rs) ranging from 0.5 to 21.0. These results included baseline separations of 11 AAs, the peaks of which were observed as the ions [AA+18C6H4+H]+. The migration order of the chiral AAs was also evaluated, and the L-AAs migrated faster than the counterpart D-AAs except for serine, threonine and methionine when using (+)-18C6H4. To reduce contamination of the ESI source by the nonvolatile chiral selector and improve the ionization efficiency in partial filling technique, the separation zone length was adjusted to 70% of the capillary, which was filled with 30 mM 18C6H4 in water. This method showed a similar separation efficiency as the typical method, and the separated AA peaks were observed as free AA ions, [AA+H]+. The optimized method provided limits of detection (LODs) ranging from 0.07 to 1.03 μg/mL and good linearity (R2 > 0.99) up to 50 μg/mL for DL-AAs. The developed method was utilized to determine DL-AAs in vinegars with a simple pretreatment process. It may be extended to sensitive AA analysis in the determination of minor enantiomeric impurities in the major component.

Enantioseparation and sensitive analysis of ofloxacin by poly(3,4-dihydroxyphenylalanine) functionalized magnetic nanoparticles-based solid phase extraction in combination with on-line concentration capillary electrophoresis

Chiral separation of low concentrations of pharmaceuticals in biofluids is a challenge task. In this study, a method is developed to enantioseparate trace amount of racemic ofloxacin in urine and bovine blood by combining magnetic solid phase extraction (MSPE) and chiral capillary electrophoresis (CE). Poly(3,4-dihydroxyphenylalanine) modified magnetic nanoparticles (polyDOPA-MNPs) are prepared and used as the adsorbents in MSPE to extract ofloxacin. The polyDOPA-MNPs are spherical, about 130 nm in diameter and the polyDOPA shell is about 3 nm. The extraction process of polyDOPA-MNPs for ofloxacin includes 2 min adsorption and 2 min desorption with the assistance of sonication. Under the optimized MSPE conditions (3 mg polyDOPA-MNPs as adsorbents, sample pH 7.0, 75% (v/v) AcOH in methanol as eluent, adsorption time 2 min, desorption time 2 min), the extraction efficiency of ofloxacin is 95%. In chiral CE, pressure-assisted field-enhanced sample injection (PA-FESI) is developed to improve the detection sensitivity of ofloxacin enantiomers. The MSPE/PA-FESI chiral CE method demonstrates a good linearity in the concentration range of 0.01–0.06 μg/mL, with a detection limit as low as 0.29 ng/mL. The feasibility of the method is verified by the successful determination of trace amounts of ofloxacin enantiomers in spiked urine and bovine blood samples.

Development of a Post-Column Liquid Chromatographic Chiral Addition Method for the Separation and Resolution of Common Mammalian Monosaccharides.

The first solely MS-based methodology for the identification and resolution of the ten common mammalian monosaccharides is presented. Based on Cooks' fixed ligand kinetic method, this technique is effective on multiple classes of monosaccharides and includes the first example of two fixed ligand combinations used in a single multiplexed experiment. Subsequently, a post-HPLC chiral addition method is used in conjunction with this newly developed MS methodology for the separation and identification of mixtures of common neutral mammalian monosaccharides. This proposed technique is able to overcome a limitation of present carbohydrate analysis methods, namely the simultaneous isomeric resolution of multiple monosaccharides in a mixture. Graphical Abstract.

The enantioselective environmental behavior and toxicological effects of pyriproxyfen in soil

We synthesized nine pyriproxyfen (PYR) metabolites and developed a chiral residual analysis method for PYR with its metabolites in five soils using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Soil degradation research showed that higher organic matter content and bigger soil particle size were conducive to the degradation of PYR and metabolites. Metabolite A 4'-OH-PYR was mainly found in five soils. PYR and metabolite A performed enantioselective degradation in soil with half-lives ranging from 2.11 d to 9.69 d and 2.80 d to 13.30 d, respectively. The activity of dehydrogenase, sucrase was inhibited and catalase activity was promoted under the disturbance of PYR. Urease was more sensitive to PYR with uncertain influences. Most soil enzymes were not restored to their initial active state after 120 d. The toxicity of metabolites to earthworms was greater than that of the parent compound PYR. This study provides the basic degradation and toxicity data of chiral pesticide PYR and its main metabolites in soil ecosystem, which is of great significance for guiding safe use and comprehensive evaluation of PYR on environmental risk.

Chiral separation and modeling of quinolones on teicoplanin macrocyclic glycopeptide antibiotics CSP.

New chiral high-performance liquid chromatography (HPLC) method for the enantiomeric resolution of quinolones is developed and described. The column used was Chirobiotic T (150×4.6mm, 5.0μm). Three mobile phases used were MeOH:ACN:Water:TEA (70:10:20:0.1%), (60:30:10:0.1%), and (50:30:20:0.1%). The flow rate of the mobile phases was 1.0mL/min with UV detection at different wavelengths. The values of retention, resolution, and separation factors ranged from 1.5 to 6.0, 1.80 to 2.25, and 2.86 to 6.0, respectively. The limit of detection and quantification ranged from 4.0 to 12ng and 40 to 52ng, respectively. The modeling studies indicated strong interactions of R-enantiomers with teicoplanin chiral selector than S-enantiomers. The supra molecular mechanism of the chiral recognition was established by modeling and chromatographic studies. It was observed that hydrogen bondings and π-π interactions are the major forces for chiral separation. The present chiral HPLC method may be used for enantiomeric resolution of quinolones in any matrices.

Chiral Purity of Crystals Using Low-Frequency Raman Spectroscopy.

The pharmaceutical industry is in need of new techniques to identify the chirality of solids due to regulatory and safety concerns regarding the biological activity of enantiomers. In this study, we present for the first time the application of low-frequency Raman spectroscopy as a new and sensitive method for analyzing the chiral purity of crystals. Using this method, we were able to identify small amounts, as low as 1 % w/w, of an enantiomer in racemic crystals. To demonstrate the capabilities of the method, we used a model system based on chiral crystals of enantiopure, racemic crystals and their mixtures in various ratios. We found that the low-frequency Raman spectra of racemic and enantiopure crystals are significantly different, reflecting the different hydrogen bond networks. Moreover, a comparison of the sensitivity of enantiomeric excess in chiral crystals to that of circular dichroism and X-ray diffraction measurements showed that low-frequency Raman attains high sensitivity comparable to chiral optical methods used for solutions. Overall, our proposed approach of using Raman spectroscopy for determining enantiomeric excess in crystals is simple, fast, and offers a high degree of chiral sensitivity.

An LC-MS/MS workflow to characterize 16 regio- and stereoisomeric trihydroxyoctadecenoic acids[S

Trihydroxyoctadecenoic acids (TriHOMEs) are linoleic acid-derived oxylipins with potential physiological relevance in inflammatory processes as well as in maintaining an intact skin barrier. Due to the high number of possible TriHOME isomers with only subtle differences in their physicochemical properties, the stereochemical analysis is challenging and usually involves a series of laborious analytical procedures. We herein report a straightforward analytical workflow that includes reversed-phase ultra-HPLC-MS/MS for rapid quantification of 9,10,13- and 9,12,13-TriHOME diastereomers and a chiral LC-MS method capable of resolving all sixteen 9,10,13-TriHOME and 9,12,13-TriHOME regio- and stereoisomers. We characterized the workflow (accuracy, 98-120%; precision, coefficient of variation ≤6.1%; limit of detection, 90-98 fg on column; linearity, R2 = 0.998) and used it for stereochemical profiling of TriHOMEs in bronchoalveolar lavage fluid (BALF) of individuals with chronic obstructive pulmonary disease (COPD). All TriHOME isomers were increased in the BALF of COPD patients relative to that of smokers (P ≤ 0.06). In both COPD patients and smokers with normal lung function, TriHOMEs with the 13(S) configuration were enantiomerically enriched relative to the corresponding 13(R) isomers, suggesting at least partial enzymatic control of TriHOME synthesis. This method will be useful for understanding the synthetic sources of these compounds and for elucidating disease mechanisms.

Simultaneous chiral impurity analysis of methamphetamine and its precursors by supercritical fluid chromatography–tandem mass spectrometry

Impurity profiling of seized illicit methamphetamine (MA) provides information on MA manufacturing methods in clandestine laboratories, and this drug intelligence supports formulation of strategies to control MA abuse. In the present study, we developed a simultaneous chiral analysis method for MA and its precursors using supercritical fluid chromatography–tandem mass spectrometry equipped with an enantioselective stationary phase. Chromatographic conditions were optimized by systematic investigation of the flow rate, temperature, back pressure, co-solvent, additive, and mobile phase composition. The ability of the developed method was evaluated using standard and authentic illicit MA. The use of a chiral selector in the stationary phase allowed for simultaneous chiral differentiation of MA and its precursors including ephedrine, norephedrine, chloropseudoephedrine, methylephedrine, dimethylamphetamine, and amphetamine. Sufficient limit of detection, repeatability of retention time, and linearity were achieved. A switching valve interfacing a chromatograph and a mass spectrometer enabled analyzing large amounts of MA directly. The application to the authentic illicit MA samples was achieved and revealed the existence of impurities, which was not detected by conventional gas chromatography–mass spectrometry. The developed supercritical fluid chromatography–tandem mass spectrometry method could be a powerful analytical tool for MA impurity profiling.

Looking for a uds¯b¯ bound state in the chiral quark model

Inspired by the report of D0 Collaboration on the state $X(5568)$ with four different flavors, a similar state, $ud\bar{s}\bar{b}$ is investigated in the present work. The advantage of looking for this state over the state $X(5568)$ with quark contents, $bu\bar{d}\bar{s}$ or $bd\bar{u}\bar{s}$, is that the $BK$ threshold is 270 MeV higher than that of $B_s \pi$, and it allows a large mass region for $ud\bar{s}\bar{b}$ to be stable. The chiral quark model and Gaussian expansion method are employed to do the calculations of four-quark states $ud\bar{s}\bar{b}$ with quantum numbers $IJ^{P}$($I=0,1;~ J=0,1,2;~P=+$). Two structures, diquark-antidiquark and meson-meson, with all possible color configurations are considered. The results indicate that energies of the tetraquark with diquark-antiquark configuration are all higher than the threshold of $BK$, but for the state of $IJ^P=00^+$ in the meson-meson structure, the energies are below the corresponding thresholds, where the color channel coupling plays an important role. The distances between two objects (quark/antiquark) show that the state is a molecular one.

Enantioselective monitoring of chiral fungicide famoxadone enantiomers in tomato, apple, and grape by chiral liquid chromatography with tandem mass spectrometry.

Famoxadone is a widely used chiral fungicide on tomato, apple, and grape. But it is still being employed as a racemic mixture without distinguishing the difference between enantiomers, which often leads to its inaccurate risk assessment. In this study, a rapid, sensitive, and reliable chiral analytical method was developed for famoxadone enantiomers by ultra high performance liquid chromatography/tandem mass spectrometry, optimal separation condition was achieved with Lux Amylose-1 column using acetonitrile/water (70:30, v/v) as mobile phase at 0.3mL/min in 6min. The average recoveries for two enantiomers in all of the matrices at three spiking levels ranged from 89.8 to 109.4%, with relative standard deviation less than 9.5%. The limits of quantification for all enantiomers in tomato, apple, and grape were not more than 4 μg/kg. And the proposed method was successfully applied to investigate the enantioselective degradation of famoxadone enantiomers in tomato, apple, and grape. The data showed that S-(+)-famoxadone was preferentially degraded comparing to the R-(-)-famoxadone in tomato, apple, and grape. The potential reasons of the enantioselective behavior were also discussed. This study could help in better understanding the environmental fate of famoxadone and the rational use of chiral pesticide in agricultural production.

Identification of Enantiomeric Byproducts During Microalgae-Mediated Transformation of Metoprolol by MS/MS Spectrum Based Networking.

Metoprolol (MPL) is a chiral β-blocker ubiquitously detected in various environments due to its low to moderate removal in wastewater treatment plants. This study was conducted to test the potential of using microalgae to degrade emerging contaminant MPL and to characterize the enantiomeric enrichment during MPL degradation by microalgae. The results showed that PO43−- P, NO3−- N and MPL could be simultaneously removed in the synthetic effluent by the targeted microalgae species, indicating microalgae were promising in wastewater treatment. Stereoselectivity was observed during MPL degradation by microalgae, with R-form enrichment. A marginal linear relationship between MPL degradation and enantiomeric enrichment was observed, implying that the enantiomeric tool, used as a quantitative indicator of biodegradation, could possibly be applied in MPL degradation by microalgae. An efficient liquid chromatograph tandem high resolution mass spectrometry (LC-HRMS/MS) chiral analytical method was developed to identify transformation products (TPs). The results showed that MS/MS spectral similarity networking could be used as a powerful tool to quickly identify unknown TPs. A total of 6 pairs of chiral TPs were identified, among which two new TPs of MPL including hydroxy{4-[2-hydroxy-3-(isopropylamino)propoxy]phenyl}acetic acid (α-HMPLA) and 4-[2-Hydroxy-3-(isopropylamino)propoxy]benzaldehyde (DMPLD) were firstly reported, and proposed transformation pathways of MPL by microalgae were given. Considering the paired TPs detected and that the degradation of the two enantiomers followed first order kinetics, the two enantiomers likely had the same degradation mechanism.

Enantioseparation and purity determination of Ondansetron by amylose based Chiral HPLC method: A Chemometric approach

Amylose based chiral HPLC method for enantioselective separation of ondansetron was developed, optimized and validated on Chiral Pak AS-3R analytical column (150 mm × 4.6 mm i.d., 3 μm). Mobile phase comprising of Acetonitrile: 0.2% Triethylamine: Methanol (15.49:30:54.51) and at a flow rate of 1.33mL/min. The separation was detected at a wavelength of 254 nm and run time was achieved within 6.0 min. Applied Central composite design (CCD) to validate the impact of three independent variables: 0.2% triethylamine (TEA), Acetonitrile (ACN) and flow rate (FL) keeping the methanol concentration constant and the responses selected were capacity factor (k1), resolution (Rs2,1), and retention time (tR2). Using this approach, a mathematical model was elucidated and response surface plots were acquired for the separation. CCD results were integrated into a multi-criteria decision-making approach in order to prevail a set of optimal experimental conditions directing to the most desirable compromise between resolution and analysis time. This method can be applied for the routine analysis.

Neutron Star Tidal Deformabilities Constrained by Nuclear Theory and Experiment

We confront observational data from gravitational wave event GW170817 with microscopic modeling of the cold neutron star equation of state. We develop and employ a Bayesian statistical framework that enables us to implement constraints on the equation of state from laboratory measurements of nuclei and state-of-the-art chiral effective field theory methods. The energy density functionals constructed from the posterior probability distributions are then used to compute consistently the neutron star equation of state from the outer crust to the inner core, assuming a composition consisting of protons, neutrons, electrons, and muons. In contrast to previous studies, we find that the 95% credibility range of predicted neutron star tidal deformabilities (136<Λ<519) for a 1.4 solar-mass neutron star is already consistent with the upper bound deduced from observations of the GW170817 event. However, we find that lower bounds on the neutron star tidal deformability will very strongly constrain microscopic models of the dense matter equation of state. We also demonstrate a strong correlation between the neutron star tidal deformability and the pressure of beta-equilibrated matter at twice saturation density.