Chiral Ligand-exchange Chromatography Research Articles

Multiple Heart-Cutting Two-Dimensional HPLC-UV Achiral–Chiral Analysis of Branched-Chain Amino Acids in Food Supplements under Environmentally Friendly Conditions

A multiple heart-cutting (mLC-LC) two-dimensional HPLC-UV achiral–chiral method for the direct analysis of branched-chain amino acids (BCAAs) in food supplements under environmentally friendly conditions was developed to cope with the very well-known limited chemoselectivity of chromatographic media for enantioselective analysis. Both achiral and chiral methods were developed in compliance with the main principles of green chromatography. The achiral analysis was performed isocratically with an optimized ion-pair reversed-phase (IP-RP) method based on a water/EtOH (95:5, v/v) mobile phase containing heptafluorobutyric acid (7 mM) as the IP agent. The achiral method was characterized by a very appreciable performance and was validated before the analysis of the real sample. High recovery values for all compounds (from 97% to 101%) were found in the interday evaluation. Additionally, low RSD% values in the long-term period were measured, in the range between 1.1% and 4.8%. Still, an LOQ value of 0.06 mg/mL was established for all compounds. The quantitative analysis of a commercial food supplement revealed that BCAAs were present in amounts very close to those declared by the producer. The enantioselective analysis was carried out through the application of the chiral ligand-exchange chromatography (CLEC) approach, using O-benzyl-(S)-serine ((S)-OBS, 0.5 mM) as the chiral selector and Cu(II) nitrate (0.25 mM) as the metal source in the eluent. Resolution and separation factor values up to 2.31 and 1.43, respectively, were obtained. The two chromatographic systems were connected through a six-port switching valve, and the developed two-dimensional mLC-LC method confirmed the absence of D-enantiomers of BCAAs in the food supplement, as reported in the manufacturer’s label.

Preparation and evaluation of an ethylenediamine dicarboxyethyl diamido-bridged bis(β-cyclodextrin)-bonded chiral stationary phase for high performance liquid chromatography

An ethylenediamine dicarboxyethyl diacetamido-bridged bis(β-cyclodextrin) was firstly synthesized through the reaction of 6-deoxy-6-amino-β-cyclodextrin (NH2-CD) with ethylenediaminetetraacetic dianhydride. Then it was bonded onto the surface of silica gel SBA-15 to obtain an ethylenediamine dicarboxyethyl diacetamido-bridged bis(β-CD)-bonded chiral stationary phase (EBCDP). The structures of the bridged bis(β-CD) and EBCDP were characterized by infrared spectroscopy, mass spectrometry, elemental analysis and thermogravimetric analysis, accordingly. The chiral chromatographic performances of EBCDP were systematically evaluated by separating 28 racemic analytes in the reversed-phase or polar organic mode, including eight flavanones, eight bolckers, five dansyl-amino acids, three DL-amino acids and four other common drugs. As a result, the relatively high enantioselectivity of EBCDP was observed in comparison with a native β-CD-CSP (CDSP). All selected analytes were separated on EBCDP, of which 20 analytes had resolutions up to baseline, 2′-hydroxyflavanone and arotinolol had resolutions up to 4.35 and 2.05 in about 30 min, respectively, whereas CDSP only separated 11 analytes with low resolutions (0.55~1.69). Moreover, EBCDP was able to utilize the complexation of the bridging linker (ethylenediamine dicarboxyethyl diamide group, EDTA-based) to realize direct separations of DL-amino acids with a mobile phase containing copper ion (Cu2+), which was similar to the chiral ligand exchange chromatography. Unlike the native cyclodextrin with small cavity (~242 Å3), the bridged bis(β-CD) combined two β-CD units with a bridging linker, having a well-organized “pseudo-cavity” as an organic whole to encapsulate more analytes, which made EBCDP have broad-spectrum applications in chiral separations.

Enantioseparations by High-Performance Liquid Chromatography Based on Chiral Ligand Exchange.

Although the first application of chiral ligand-exchange chromatography (CLEC) in HPLC dates back to late 1960s, this enantioselective strategy still represents the elective choice for the direct analysis of compounds endowed with chelating moieties. As a specific feature of the CLEC mechanism, the interaction between the chiral selector and the enantiomer does not take place in direct contact. Indeed, it is mediated by a central metal ion that, acting as a Lewis acid, simultaneously coordinates the two species, selector and analyte, through the activation of dative bonds. As a consequence, two diastereomeric mixed ternary complexes are generated in the column, ultimately leading to the stereoisomeric discrimination. CLEC applications can be carried out both with the chiral selector included in the mobile phase (chiral mobile phase, CMP), or as a part of the stationary phase. In the latter case, the chiral selector can be either covalently immobilized onto a solid support (bonded CSP, B-CSP) or physically adsorbed onto a conventional packing material, coated chiral stationary phase (C-CSP).In this chapter, a selection of CLEC applications with CMP- and C-CSP-based chiral systems is presented.

Last ten years (2008-2018) of chiral ligand-exchange chromatography in HPLC: An updated review.

Chiral ligand-exchange chromatography is one of the elective strategies for the direct enantioresolution of small chelating compounds: amino acids, diamines, amino alcohols, diols, small peptides, etc. Unlike other methods, the interaction between chiral selector and analyte enantiomers is mediated by a cation, thus producing diastereomeric ternary complexes. Two main approaches are conventionally applied in chiral ligand-exchange chromatography. The first relies upon chiral stationary phases where the chiral selector is either covalently immobilized or physically adsorbed onto suitable packing materials (coated phases). In the second approach, chiral molecules are added to the eluent, thus generating chiral eluent systems. Among the advantages of chiral ligand-exchange chromatography, the generation of UV/vis-active metal complexes, and the use of commercially available or easy-to-synthesize chiral selectors, in combination to rather inexpensive achiral columns for coated phases and chiral eluents, are noteworthy. Besides amino acids and amino alcohols, other species have proven suitable for chiral ligand-exchange chromatography applications. Recently, the use of either chiral ionic liquids or micellar liquid chromatography systems as well as the successful off-column formation of diastereomeric complexes have expanded the selectivity profiles and application fields. All of these issues are touched in the review, shedding light to the contributions appeared in the last decade.

C-Methylation Catalyzed by Fom3, a Cobalamin-Dependent Radical S-adenosyl-l-methionine Enzyme in Fosfomycin Biosynthesis, Proceeds with Inversion of Configuration.

Fom3, a cobalamin-dependent radical S-adenosyl-l-methionine (SAM) methyltransferase, catalyzes C-methylation at the C2 position of cytidylylated 2-hydroxyethylphosphonate (HEP-CMP) to afford cytidylylated 2-hydroxypropylphosphonate (HPP-CMP) in fosfomycin biosynthesis. In this study, the Fom3 reaction product HPP-CMP was reanalyzed by chiral ligand exchange chromatography to confirm its stereochemistry. The Fom3 methylation product was found to be ( S)-HPP-CMP only, indicating that the stereochemistry of the C-methylation catalyzed by Fom3 is ( S)-selective. In addition, Fom3 reaction was performed with ( S)-[2-2H1]HEP-CMP and ( R)-[2-2H1]HEP-CMP to elucidate the stereoselectivity during the abstraction of the hydrogen atom from C2 of HEP-CMP. Liquid chromatography-electrospray ionization mass spectrometry analysis of the 5'-deoxyadenosine produced showed that the 2H atom of ( R)-[2-2H1]HEP-CMP was incorporated into 5'-deoxyadenosine but that from ( S)-[2-2H1]HEP-CMP was not. Retention of the 2H atom of ( S)-[2-2H1]HEP-CMP in HPP-CMP was also observed. These results indicate that the 5'-deoxyadenosyl radical stereoselectively abstracts the pro-R hydrogen atom at the C2 position of HEP-CMP and the substrate radical intermediate reacts with the methyl group on cobalamin that is located on the opposite side of the substrate from SAM. Consequently, it was clarified that the C-methylation catalyzed by Fom3 proceeds with inversion of configuration.

Direct HPLC separation of carnosine enantiomers with two chiral stationary phases based on penicillamine and teicoplanin derivatives.

Carnosine is present in high concentrations in specific human tissues such as the skeletal muscle, and among its biological functions, the remarkable scavenging activity toward reactive carbonyl species is noteworthy. Although the two enantiomers show almost identical scavenging reactivity toward reactive carbonyl species, only d-carnosine is poorly adsorbed at the gastrointestinal level and is stable in human plasma. Direct methods for the enantioselective analysis of carnosine are still missing even though they could find more effective applications in the analysis of complex matrices. In the present study, the use of two different chiral stationary phases is presented. A chiral ligand-exchange chromatography stationary phase based on N,S-dioctyl-d-penicillamine resulted in the direct enantioseparation of carnosine. Indeed, running the analysis at 25°C and 1.0mL/min with a 1.5mM copper(II) sulfate concentration allowed us to obtain separation and resolution factors of 3.37 and 12.34, respectively. However, the use of a copper(II)-containing eluent renders it hardly compatible with mass spectrometry detectors. With the teicoplanin-based stationary phase, a mass spectrometry compatible method was successfully developed. Indeed, a water/methanol 60:40 v/v pH 3.1 eluent flowed at 1.0mL/min and with a 25°C column temperature produced separation and resolution factors of 2.60 and 4.16, respectively.

Development of a New Method Based on Chiral Ligand-Exchange Chromatography for the Enantioseparation of Propranolol.

A new chromatographic procedure was proposed for the separation of propranolol (PRN) enantiomers based upon enantioselective chiral ligand-exchange chromatography. The separation was carried out on a short C8 column leading to considerably short separation time. L-alanine and Cu2+ were applied as chiral selector and central bivalent complexing ion, respectively. It was found that the kind of copper salt could influence the enantioseparation efficiency. The separation on the C8 stationary phase was more efficient than that on the C18 column. It was shown that the pH of mobile phase, organic modifier content of mobile phase, mole ratio of chiral ligand to bivalent ion and Cu (L-alanine) 2 concentration in the mobile phase were important in enantioseparation efficiency. Water/methanol (70:30) mixture containing L-alanine-Cu2+ (7:1) was found to be the best mobile phase condition for PRN enantioseparation. All effective parameters were optimized in order to improve the separation efficiency. The optimized HPLC method was utilized for analysis of propranolol enantiomers in spiked human blood plasma samples.

Cyclopropyl-containing sulfonyl amino acids: Exploring the enantioseparation through chiral ligand-exchange chromatography

In the scope of a broader study focused on glutamate receptors regulators, we have been engaged in synthesis, analysis and pharmacological characterization of rigid analogues of glutamic acid. These compounds exhibited the bioisosteric replacement of the distal carboxylic group with the sulfonic one. Besides the sophisticated synthetic approach, we targeted preparation of a series of cyclopropyl-containing sulfonyl amino acids and development of a chromatographic enantioselective method suitable for distinguishing and quantifying the resulting isomers. Due to chelating ability, the chiral ligand-exchange chromatography (CLEC) was used for diastereo- and enantioseparation of the synthesized compounds. The CLEC-based enantioseparation was achieved by using a chiral mobile phase (CMP) system with N,N-dimethyl-(S)-phenylalanine [(S)-DMP] as the chiral selector. Only one of the investigated enantiomeric pairs was undiscriminated with the employed CLEC–CMP system which, very importantly, produced the simultaneous diastereo- and enantioseparation of two compounds of the series. Furthermore, the large α and RS values computed for three enantiomer pairs could be a good basis for a successful scale-up to a semi-preparative level.

Chiral Ligand-Exchange Chromatography of Pharmaceutical Compounds on Dynamically Coated (Home-Made) Stationary Phases.

It is well known for several decades that the two enantiomeric forms of a chiral compound can have very different effects on the human body. For this reason the synthesis or extraction from a natural source of a potential new drug, as well as its marketing, require a careful control of its optical purity. Chromatographic techniques can respond extremely well to this need, both in the analytical and in the preparative field. Among the several methods developed for this purpose, one of the first and of the most effective is the Chiral Ligand-Exchange Chromatography, which is based on the stability difference between the metallic diastereomeric complexes containing one or the other of the two enantiomers to be separated and a suitable chiral selector. This method has been effectively used for resolving racemic mixtures of products of biomedical and/or pharmacological interest, such as α - and β-amino acids either proteinogenic or non-proteinogenic, oligopeptides, amino alcohols or β-blockers. All these substances are linked together by their ability to bind metal ions, the most widely used of which is Cu(II). The chiral selector can be a component of either the mobile or the stationary phase, to which it can be either chemically bonded or dynamically adsorbed. The latter method has several advantages of convenience and, above all, cheapness. The preparation of dynamically-coated chiral stationary phases for Ligand-Exchange Chromatography has produced a large number of applications, the main of which, both in TLC and in HPLC, are reviewed below.

Preparation and Characterization of Cellulose Membrane Modified with β-Cyclodextrin for Chiral Separation

In this study, it is first time to obtain a complete separation of using β-cyclodextrin (β-CD) modified cellulose microfiltration membrane. Commercially cellulose membrane with the pore diameter of 0.22μm was functionalized with β-cyclodextrin (β-CD) by aldolization at the solid–liquid interface. Filtration experiments were carried out using a dead-end filtration cell holding a flat sheet membrane with effective area of 36 mm2. Aqueous solution of racemic tryptophan (0.05g/L, 150 mL) was forced to permeate through the cellulose membrane immobilized by β-CD at a flow rate of 0.1 ml/min through the membrane microdevice. Chiral ligand exchange chromatography was used to determine the concentration and ratio of D- and L-tryptophan in the filtrate. A complete separation of racemic tryptophan can be obtained by using this novel composite membrane-based separation system. In addition, a multi-stage filtration separation was applied in order to obtain higher permselectivity. The objective of this study is to obtain an easy prepared chiral membrane with good reproducibility and can be applied to a variety of chiral separations.

Chiral ligand-exchange chromatography with Cinchona alkaloids. Exploring experimental conditions for enantioseparation of α-amino acids

The natural Cinchona alkaloid quinidine as chiral selector in chiral ligand-exchange chromatography was systematically studied. Chromatographic conditions for enantioseparation of twenty α-amino acids were first time studied by changing mobile phase parameters such as pH, concentration of organic solvent, type of salt, ligand to metal ratio and also column temperature. Maximum retention and enantioselectivity factors were observed at the region close to pH=8, since the tertiary amine (the quinuclidinic nitrogen) of the quinidine is protonated only in a small degree, and therefore is available for the chelate formation. Additionally at this pH value there is no other competing ligand for complex the metallic cation. The thermodynamic transfer parameters of the enantiomers from the mobile to the stationary phase from van't Hoff plots within the range of 10–35°C were estimated. Thus, the differences in the transfer enthalpy Δ(ΔH), and transfer entropy Δ(ΔS) enabled an investigation of the origin of the differences in interaction energies Δ(ΔG). Finally, the molecular geometry of the formed diastereomeric complexes was modelled and energetic differences between both compounds were calculated by a semi empirical method.

Chiral resolution of salbutamol in plasma sample by a new chiral ligand-exchange chromatography method after its extraction with nano-sized imprinted polymer

A new chromatographic procedure, based upon chiral ligand-exchange principal, was developed for the resolution of salbutamol enantiomers. The separation was carried out on a C18 column. (l)-Alanine and Cu(2+) were applied as chiral resolving agent and complexing ion, respectively. The kind of copper salt had definitive effect on the enantioseparation. Density functional theory (DFT) was used to substantiate the effect of various anions, accompanying Cu(2+), on the formation of ternary complexes, assumed to be created during separation process. The DFT results showed that the anion kind had huge effect on the stability difference between two corresponding diastereomeric complexes and their chemical structures. It was shown that the extent of participation of the chiral selector in the ternary diastereomeric complexes formation was managed by the anion kind, affecting thus the enantioseparation efficiency of the developed method. Water/methanol (70:30) mixture containing (l)-alanine-Cu(2+) (4:1) was found to be the best mobile phase for salbutamol enantioseparation. In order to analyze sulbutamol enantiomers in plasma samples, racemic salbutamol was first extracted from the samples via nano-sized salbutamol-imprinted polymer and then enantioseparated by the developed method.

Separation of Ofloxacin and Its Six Related Substances Enantiomers by Chiral Ligand-Exchange Chromatography.

A chiral ligand-exchange high-performance liquid chromatography method was developed for the enantioseparation of ofloxacin and its six related substances termed impurities A, B, C, D, E, and F. The separation was performed on a conventional C18 column. Different organic modifiers, copper salts, amino acids, the ratio of Cu(2+) to amino acid, pH of aqueous phase, and column temperature were optimized. The optimal mobile phase conditions were methanol-water systems consisting of 5 mmol/L copper sulfate and 10 mmol/L L-isoleucine (L-Ile). Under such conditions, good enantioseparation of ofloxacin and impurities A, C, E, and F could be observed with resolutions (RS ) of 3.54, 1.97, 3.21, 3.50, and 2.12, respectively. On the relationship between the thermodynamic parameters and structures of analytes, the mechanism of chiral recognition was investigated. It was concluded that ofloxacin and impurities A, C, E, and F were all enthalpically driven enantioseparation and that low column temperature was beneficial to enantioseparation. Furthermore, the structure-separation relationship of these analytes is also discussed.

Modeling the retention mechanism for high-performance liquid chromatography with a chiral ligand mobile phase and enantioseparation of mandelic acid derivatives.

The chromatographic retention mechanism describing relationship between retention factor and concentration of Cu(2+) (l-phenylalanine)2 using chiral ligand mobile phase was investigated and eight mandelic acid derivatives were enantioseparated by chiral ligand exchange chromatography. The relationship between retention factor and concentration of the Cu(2+) (l-phenylalanine)2 complex was proven to be in conformity with chromatographic retention mechanism in which chiral discrimination occurred both in mobile and stationary phase. Different copper(II) salts, chiral ligands, organic modifier, pH of aqueous phase, and conventional temperature on retention behavior were optimized. Eight racemates were successfully enantioseparated on a common reversed-phase column with an optimized mobile phase composed of 6 mmol/L of l-phenylalanine or N,N-dimethyl-l-phenylalanine and 3 mmol/Lof copper(II) acetate or copper(II) sulfate aqueous solution and methanol.

Use of an o-Benzyl-(S)-Serine Containing Eluent for the Efficient Ligand-Exchange Chromatography-Based Enantioseparation of Constrained Glutamate Receptor Ligands

Four dihydroisoxazole prolines and four dihydroisoxazole cyclopentane derivatives were submitted to chiral ligand-exchange chromatographic analysis in the presence of O-benzyl-(S)-serine, as the chiral mobile phase additive to the eluent. The 1.0 mM O-benzyl-(S)-serine and 0.5 mM Cu(NO3)2 eluent flowed at 1.0 mL/min through a conventional octadecylsilica-based stationary phase maintained at 25°C and provided excellent levels of enantioselectivity and resolution for all the species. By using enantiomers as model compounds, the method was validated revealing that the mixed ternary diastereomeric eluates present slightly different spectroscopic properties. The selected chiral ligand-exchange chromatography method was applied for semi-preparative enantioisolation that allowed the establishment of the k(−) < k(+) enantiomeric elution order.

Indirect chiral ligand exchange chromatography for enantioseparation: a modification of conventional techniques

Indirect chiral ligand exchange chromatography (ICLEC) was facilitated by substituting chiral ligands from the bidentate Cu(II) complex via racemic analytes in reaction vial. In-vial ligand exchange does not require the potentially harmful Cu(II)-containing mobile phase for HPLC separation, as it facilitates better pH control for improved complexation of enantiomers with the chiral Cu(II)–ligand complex. Resulting Cu(II)–analyte–ligand complexes have higher stability constants and show better chromatographic resolution than conventional (CLEC). The new technique also eliminates background noise, a problem with conventional CLEC due to higher concentrations of Cu-containing selector due to the Cu-free mobile phase. The new technique was found successful in determining enantiomeric purity of drugs in pharmaceutical formulations. The technique can be regarded as a novel surrogate for conventional CLEC and has the potential for biological and pharmaceutical applications.

Isoleucine ionic liquids as additives to separate mandelic acid and their derivative enantiomers by HPLC

In this study, functional isoleucine ionic liquids were synthesized and used as novel chiral ligands coordinated with Cu(ii) in chiral ligand exchange chromatography.

Combined monodimensional chromatographic approaches to monitor the presence of d-amino acids in cheese

The presence of d-amino acids (d-AAs) as a consequence of natural or artificial interventions such as ageing, microorganism action, preservative and conservative processes (alkali or heat treatment), is a scarcely treated aspect from the scientific community. It is also fully documented that even a minor degree of racemisation on the proteins' AAs is the cause of a reduced digestion of such proteins. Besides interfering with the regular metabolism of l-AAs, d-AAs can also contribute to the development of pathological conditions in humans. So far, nearly all the most important chromatographic techniques were applied to quantify d-AAs in foodstuffs. However, most of them rely upon pre- or post-column derivatization procedures, often combined with sophisticated analytical equipments. Differently, in this paper we propose an easy-to-set up combination of monodimensional chromatographic methods to monitor the variation of the d-Ala, d-Asp and d-Glu content in two commercially available Spanish cheese samples prepared from the same milk mixture and characterized by a different maturity time: no ripening and six months ripening. After the free amino acid mixture was extracted from the two cheese samples, an ion-pairing RP-HPLC achiral protocol was firstly optimized with the objective to avail of a method enabling the complete distinction of Ala, Asp, and Glu from all the other aminoacidic species in the two extracts. An ion-exchange-based chromatographic method was also optimized, thus allowing a profitable fractionation of the two aminoacidic mixtures. With such a procedure, less complex samples to be analyzed with a chiral ligand-exchange chromatography (CLEC) stationary phase based on S-trityl-l-cysteine (l-STC) units were obtained.The optimized CLEC conditions were then applied to the previously identified Ala, Asp and Glu containing fractions as well as to those including all the remaining species. For all the three compounds the enantiomeric excess (ee) was found to decrease passing from the ripened to the fresh cheese. As expected, the largest difference was found for Ala (ee value from 83.0% down to 20.5%), followed progressively by Asp (ee value from 90.5 to 75.0%) and Glu (ee value from 99.0 to 91.8%).

Enantioseparation of α-amino acids by means of Cinchona alkaloids as selectors in chiral ligand-exchange chromatography

A conventional nonchiral column was used for the enantioseparation of several racemic α-amino acids (native and derivatized) through the use of Cinchona alkaloids as chiral selectors along with Cu(II) ions in chiral ligand-exchange chromatography. The mobile phase composition (i.e., the organic modifier content and pH) was studied in order to modulate retention and enantioselectivity. Good enantioseparation of many amino acids was obtained using equimolar amounts of Cu(II) and either cinchonidine, quinine or quinidine as chiral selectors in the mobile phase. The molecular geometry of the diastereomeric complexes formed was modeled and energetic differences between both compounds were calculated by methods based on semi-empirical force-field. Good correlations were obtained between experimental enantioselectivity factors and calculated energetic differences.

Chiral mobile phase in ligand-exchange chromatography of amino acids: Exploring the copper(II) salt anion effect with a computational approach

With the use of a chiral ligand-exchange chromatography (CLEC) system operating with the O-benzyl-(S)-serine [(S)-OBS] [1,2] as the chiral mobile phase (CMP) additive to the eluent, the effect of the copper(II) anion type on retention (k) and separation (α) factors was evaluated, by rationally changing the following experimental conditions: salt concentration and temperature. The CLEC–CMP analysis was carried out on ten amino acidic racemates and with nine different cupric salts. While the group of analytes comprised both aliphatic (leucine, isoleucine, nor-leucine, proline, valine, nor-valine, and α-methyl-valine) and aromatic (1-aminoindan-1,5-dicarboxylic acid, phenylglycine, and tyrosine) species, representative organic (formate, methanesulfonate, and trifluoroacetate) and inorganic (bromide, chloride, fluoride, nitrate, perchlorate, and sulfate) Cu(II) salts were selected as the metal source into the eluent. This route of investigation was pursued with the aim of identifying analogies among the employed Cu(II) salts, by observing the variation profile of the selected chromatographic parameters, upon a change of the above experimental conditions. All the data were collected and analyzed through a statistical approach (PCA and k-means clustering) that revealed the presence of two behavioral classes of cupric salts, sharing the same variation profile for k and α values. Interestingly, this clustering can be explained in terms of ESP (electrostatic surface potential) balance (ESPbal) values, obtained by an ab initio calculation operated on the cupric salts. The results of this appraisal could aid the rational choice of the most suitable eluent system, to succeed in the enantioseparation of difficult-to-resolve compounds, along with the eventual scale-up to a semi-preparative level.