Ligand-exchange Chromatography Research Articles

Two-Dimensional Transport Dispersive Model for Separating Glycolic Acid and NaCl with Ligand Exchange Chromatography

Two-Dimensional Transport Dispersive Model for Separating Glycolic Acid and NaCl with Ligand Exchange Chromatography

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.

Highly sensitive ligand exchange chromatographic determination of apiose in plant biomass

Apiose is an unusual branched-chain pentose monosaccharide which does not occur naturally in a free form and included in the structure of some pectins and apioglycosides possessing high biological activity and having significant potential for use in pharmacology and cosmetic industry. The control of the chemical composition of plant raw materials and related products requires reliable analytical methods for determining apiose. In the present study a ligand exchange chromatography on a stationary phase bearing lead cations in combination with post-column derivatization by L-arginine and fluorescence detection was proposed for the highly sensitive and selective quantification of apiose. The optimized sample preparation procedure involves microscale two-stage hydrolysis with 72% and 0.5 M sulfuric acid at 30 °C and 100 °C, respectively. The developed and validated simple, robust and low-cost analytical method ensures limit of detection of 15 μg L–1 in liquid samples and 5 μg g−1 in plant material with a linear range comprising up to four orders of magnitude. It was successfully tested in the analyses of pharmaceutical preparations of the eelgrass pectins, parsley and fireweed biomass.

One-Step Isolation of Protein C-Terminal Peptides from V8 Protease-Digested Proteins by Metal Oxide-Based Ligand-Exchange Chromatography

We have developed a one-step method to isolate protein C-terminal peptides from V8 protease-digested proteins by metal oxide-based ligand-exchange (MOLEX) chromatography. V8 protease cleaves the C-terminal side of Asp and Glu, affording a digested peptide with two carboxy groups at the C-terminus, whereas the protein C-terminal peptide has only one α-carboxy group. In MOLEX chromatography, a stable chelate is formed between dicarboxylates and metal atoms, so that the nonterminal (i.e., internal) peptide is retained, whereas the protein C-terminal peptide flows through the MOLEX column. After the optimization of the MOLEX chromatographic conditions, 1619 protein C-termini were identified from 30 μg of peptides (10 μg each, in triplicate) derived from human HeLa cells by means of nanoLC/MS/MS. When the MOLEX-isolated sample from 200 μg of HeLa peptides was further divided into six fractions by high-pH reversed-phase liquid chromatography (LC) prior to nanoLC/MS/MS, 2203 protein C-termini were identified with less than 3% contamination with internal peptides. We believe that this is the largest coverage with the highest purity reported to date in human protein C-terminomics. This fast, simple, sensitive, and selective method to isolate protein C-terminal peptides should be useful for profiling protein C-termini on a proteome-wide scale.

A plausible prebiotic selection of ribose for RNA - formation, dynamic isolation, and nucleotide synthesis based on metal-doped-clays

<h2>Summary</h2> The natural selection of ribose as the exclusive sugar for RNA has been one of the greatest puzzles in the origins of life. Here, we present a plausible solution to this problem that circumvents the limitations of previous hypotheses, inspired by the unique retention properties of ribose in ligand-exchange chromatography. It is discovered that metal cations and clay minerals have a synergistic effect to selectively bind to ribose against other monosaccharides. Excitingly, ribose can be continuously and highly selectively enriched on metal-doped-clays (MDCs) from a complex Formose mixture, which could be the origin of its rise to prominence. Prebiotic scenarios of the dynamic isolation of ribose from sugar mixture are simulated with microflow technology. The MDC-retained ribose exhibits long-term stability for the upcoming nucleosidation reactions. Computational studies help elucidate the unique stability of the ribose-MDC complex. MDC also promotes the 5′-phosphorylation of nucleosides with greater yield and selectivity than reported.

Optimization of a Ligand Exchange Chromatography Method for the Enantioselective Separation of Levofloxacin and Its Chiral Impurity.

Levofloxacin is a third-generation fluoroquinolone that has several advantages over its (R) ofloxacin isomer. It is used to treat different types of infection, including urinary infection and prostatitis. A new HPLC method for the enantioselective separation of levofloxacin and its chiral impurity was developed and validated to improve the separation of the enantiomers of levofloxacin [impurity(R) and active principle (S)] by increasing the value of the resolution between the eutomer and the distomer. Chromatographic separation was performed on a Prodigy ODS -2, 5 µm 4.6 × 150 mm column, with a gradient of buffer solution and methanol (80:20, v/v). A Box-Behnken design was considered when optimizing the enantioseparation involving the effects of many factors such as the concentration of d-phenylalanine, the pH of the buffer, the percentage of organic modifier in the mobile phase, the flow rate, the temperature of the column, and the type of column. Chiral separation was achieved with an optimal resolution of 3.8. The method was successfully validated following the International Conference on Harmonization Q2 (R1) guideline, fulfilling the acceptance criteria for selectivity [no interference in the retention time of (S) levofloxacin and (R) levofloxacin], linearity (r ≥0.999 in the range 1.25-3.75 µg/mL for all enantiomers), and precision (RSD <2%). Accuracy was assessed by the application of the analytical method to an analyte of known purity, providing evidence for the usefulness of this monitoring system. The method was successfully used for the determination of levofloxacin impurity in raw material and pharmaceutical dosage forms. The following method is accurate and robust to quantify and characterize the presence of levofloxacin impurity in raw material for pharmaceutical compounds.

Front Cover: Analysis of underivatized alditols in traditional Chinese medicines by liquid chromatography–tandem mass spectrometry based on ligand exchange chromatography

Front Cover: Analysis of underivatized alditols in traditional Chinese medicines by liquid chromatography–tandem mass spectrometry based on ligand exchange chromatography

Isolation and characterization of sulfur-containing compounds by positive-ion electrospray ionization and online HPLC/Atmospheric pressure chemical ionization coupled to Orbitrap mass spectrometry

Extraction and fractionation of sulfur-containing compounds from vacuum gas oil were carried out by using non-reactive methods such as ligand exchange chromatography (LEC) on solid-phase extraction cartridges and a molecularly imprinted polymer (MIP). All fractions obtained by these methods were methylated, and sulfur compounds (S1) were analyzed as methyl sulfonium ions [M + CH3]+ by positive-ion ESI Orbitrap mass spectrometry. LEC on strong cation exchange sorbents modified with Ag+ (SCX-Ag+) and Cu2+ (SCX-Cu2+) cations isolate sulfides and thiophenes through complexation of the sulfur atoms with the metal cations. In contrast, LEC on the stationary silica phase modified with Ag+ cations (Si-Ag+) was selective of sulfur species (S1) with higher DBE values near the planar limit. Interestingly, LEC/SCX-Cu2+ allowed the extraction of disulfides and other sulfur compound classes such as OS and OS2 detected as [M + H]+. Among LEC separations, the Si-Ag+ provides the largest number of molecular assignments (565) of sulfur species (S1) followed by SCX-Ag+ and SCX-Cu2+ with 463 and 408 formulae, respectively. On the other hand, a polymer with dibenzothiophene molecules imprinted afforded the highest number (820) of molecular formulae (CcHhS1) with a wider DBE ~1–20 and carbon number ~30–70 in comparison with the species (S1) eluted by LEC methods. A further separation by online HPCL/APCI (+) Orbitrap MS of the fractions obtained by SCX-Ag+ allows detecting sulfur compounds with DBE values up to 23. This contribution provides alternatives for the isolation and fractionation of sulfur-containing compounds that may be extended to heavy crude oil and asphaltenes.

Analysis of underivatized alditols in traditional Chinese medicines by liquid chromatography–tandem mass spectrometry based on ligand exchange chromatography

AbstractAlditols are potential chemical markers in traditional Chinese medicine for quality control purpose. For example, mannitol is one of the major compounds in Cordyceps sinensis. Galactitol is the proposed marker compound of formulated oral traditional Chinese medicine liquid “Fufangfufangteng Heji.” Development of a highly selective analytical method for unambiguous identification of selected alditols is of prime importance to assure the quality of these valued and precious traditional Chinese medicines. In this study, a liquid chromatography–tandem mass spectrometry method based on ligand exchange chromatography was developed to analyze and identify underivatized alditols such as galactitol, mannitol and sorbitol in different traditional Chinese medicines. The limits of detection of these alditols ranged from 0.7 to 1.4 mg/kg. The limits of quantification of these alditols ranged from 2.3 to 4.6 mg/kg. The method has been applied in the detection of alditols in various samples such as Cordyceps sinensis, Cordyceps militaris, and concentrated granule of Radix Celastrus orbiculatus. The results indicated that several underivatized alditols could be identified rapidly and unambiguously by liquid chromatography–tandem mass spectrometry on ligand exchange chromatography column.

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.

Xylitol Separation from a Polyol Mixture Using Lanthanide Ion-loaded Resins.

Xylitol separation from a polyol mixture of the byproducts from bioethanol production processes was performed by liquid chromatography using short columns packed with lanthanide ion-loaded ion-exchange resins. Xylitol was successfully separated with sufficiently high resolution using adsorbents with medium rare-earth metal ions, such as Nd3+ and Sm3+. The adsorbents' specific nature is explained by the so-called "gadolinium break," which is known as a discontinuous behavior of thermodynamic parameters in complexation of the lanthanide series. From the observed behavior, the optimum lanthanide ions could be chosen to prepare appropriate adsorbents for ligand-exchange chromatography of given polyol mixtures.

Equilibrium of aliphatic amino acids on ion exchangers forming complexes in the presence of copper (II) and nickel (II) cations

The task of isolating and separating amino acids from aqueous solutions exists in various industries. The traditional method of isolation is ligand exchange chromatography. When choosing a cation for ligand-binding chromatography based on its binding strength with the ion exchanger, often used as a sulfonated polystyrene ion exchanger keeps the copper (II) firmly enough, and therefore, it is easily replaced by other cations. Chelating ion exchangers charge cations of copper (II), which hold these ions firmly enough. In this case, separating a mixture of substances, it is due to differences in the constants of complexation agents and complexes distribution coefficients. The study of the interaction of amino acids with the aliphatic carboxylic acid, the exchange of phosphoric acid cations and the amino carboxylic and amino phosphonic polyampholytes has shown a significant effect of the pH of the medium on the nature of the sorption equilibria. Under certain conditions, in the phase of the ion exchanger in the form of complexing metal cations, the formation of new sorption centers is possible, which occur upon sorption of amino acids in the formation of mixed ligand compounds: the sorbent complex may simultaneously comprise amino acids and attached functional groups of the sorbent as ligands. The influence of the hydrogen index of the medium primarily affects the change in the nature of the formed complex compounds in the sorbent phase and the equilibrium solution and the ratio of their stability constants. If the stability constant of the ion exchanger complex is higher than the stability constant of the compound with a low molecular weight ligand, then the sorbed copper cations interact with incoming methionine ions without breaking the metal – functional group of the ion exchanger coordination bond. If the ratio of stability constants is the opposite, then the predominant elution of copper (II) cations occurs with the formation of complex compounds with an amino acid in an aqueous solution.

P.113 The nitric oxide donors nebivolol and S-nitroso-glutathione decrease the spontaneous motor behaviour in rats

In search for new enantioselectivity profiles, the N-decyl-S-trityl-(R)-cysteine [C10-(R)-STC] was synthesized through a one-step procedure and then hydrophobically adsorbed onto an octadecylsilica surface to generate a stable chiral stationary phase for ligand-exchange chromatography (CLEC-CSP) applications. The CLEC analysis was carried out on underivatized amino acids, by using a Cu(II) sulphate (1.0 mM) containing aqueous eluent system. Most of the analysed compounds (34 out of 45) were enantiodiscriminated by the C10-(R)-STC-based CSP, with resolution factor (RS) values up to 8.86. Conformationally rigid and hydrophobic ligands often experienced the largest enantioselectivity effects. A high loadability emerged from the analysis of rac-NorVal (selected as prototype test compound): up to 20 mg/mL were efficiently enantioseparated with the CLEC-CSP. Two in-line hand-made cartridges filled with a strong cation-exchange resin allowed the effective catching of Cu(II) ions after the semi-preparative enantioseparation. The quantitative recovery of the rac-NorVal enantiomers was made possible by flowing through the cartridge a 5% (v) ammonia solution. The CLEC phase proved successful in the enantioselective analysis of a commercially available (S)-Leu containing tablet. Furthermore, in order to understand the molecular basis for a successful use of the C10-(R)-STC-based CLEC system, a descriptive structure-separation relationship study was performed. As a result, all compounds with a MEAN-QPlogS (a hydrophilicity descriptor) value lower than 0.373 can be most likely enantioseparated with the CLEC system under investigation. In the work, the numerous aspects complying with the principles of green chromatography are highlighted and discussed.

Cocoa-specific flavor components and their peptide precursors.

Essential precursors of the cocoa-specific roasting-flavor notes were formed during proteolysis of the cocoa vicilin-class(7S) globulin by a mixture of cocoa aspartic protease and carboxypeptidase. These could be partially purified by ligand-exchange chromatography. Many constituents of this peptide fraction were destroyed by post-treatment with pepsin, but the cocoa-specific flavor-precursor peptides were largely resistant against pepsin treatment. However, these peptides were not generated when the cocoa vicilin-class(7S) globulin was digested with a mixture of pepsin and carboxypeptidase. By nano-liquid chromatography mass spectrometry, the peptide composition of these peptide fractions were compared in order to identify the putative precursors of the cocoa-specific flavor components. These peptides were assigned to five regions of the cocoa vicilin-class(7S) globulin. Analyzing the roasting products of the different protein fractions by headspace solid-phase microextraction, followed by gas chromatography mass spectrometry, eight volatile compounds were detected, whose occurrence correlated with the sensory detection of cocoa-specific flavor notes.

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.

Development and validation of a copper ligand-exchange chromatographic method for the estimation of D-lactic acid in Ringer-lactate solution

In this study, the chromatographic conditions for separation and determination of L- and D-lactic acid enantiomers by copper ligand exchange chromatography have been examined and optimized statistically using a response surface methodology (RSM). The chromatographic variables: copper sulfate, acetic acid and organic modifier were screened by operating a 2-level full factorial design (FFD). The significant effect of independent chromatographic variables was analyzed using the analysis of variance (ANOVA). Variables proved significant (p < 0.05) were cautiously tuned using RSM with a face-centered central composite design. Moreover, a D-optimality design was employed to minimize the variation in the regression coefficients of the fitted model. The proposed model represented an excellent example of fulfilling the efficiency of factorial designs in optimizing the chromatographic conditions and maximizing the output. The chromatographic separation was achieved on Supelco Astec CLC-D chiral bidentate ligand (5.0 µm, 150.0 × 4.6 mm). The isocratic mobile phase composition was 7 mM anhydrous copper sulfate in 1.0 mM acetic acid containing 4% methanol. A photodiode array detector was used to determine the optimal detection wavelength, which was at 236 nm. A linear calibration curve was obtained in the range of 30.0–3600 µg mL−1 with a high value for the coefficient of determination (R2 ≥ 0.999). The optimized method has been successfully applied to the determination of lactate in the commercial Ringer-lactate solution for injection. The results obtained were in excellent agreement with the label claim with no interference from other additives commonly co-formulated with the drug. Intra- and inter-day precision, detection and quantification limits, as well as percent coefficient of variation, have been estimated according to ICH guidelines for assessment of analytical procedures.

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.

Separation of levoglucosan supramers by high performance liquid chromatography

Analysis of levoglucosan by ligand-exchange chromatography with UV detection showed deviations from the Bouguer—Lambert—Beer law. Their character allowed us to suggest that the deviations are caused by the formation of levoglucosan supramers in the eluent.