Ligand-exchange High-performance Liquid Chromatography Research Articles

Simultaneous determination of ofloxacin enantiomers in water by high performance liquid chromatography

A simple and rapid method was developed for the analysis of ofloxacin enantiomers by chiral ligand exchange-high performance liquid chromatography (CLE-HPLC). In addition, the influences of common cations (Ca2+, Mg2+, Fe3+, Zn2+) and the content of humic acid (HA) on the separation were investigated. The separation was carried out on a C18 column (25 cm×0.46 cm, 5 μm). The mobile phase was 20% (v/v) methanol aqueous solution containing 4 mmol/L L-isoleucine (as ligand) and 3 mmol/L CuSO4. The pH of the mobile phase was 4.5, and the flow rate was 1.0 mL/min. The column temperature was 40℃, and the detection wavelength was 293 nm. The ofloxacin enantiomers (ofloxacin and levofloxacin) were separated within 18 min and the resolution (R) was 2.70. The results showed that metal cations and HA had no significant influence on the resolution of ofloxacin enantiomers. However, the measured peak area of ofloxacin enantiomers decreased particularly in presence of Fe3+ and on increasing HA content. The proposed method can rapidly and efficiently determine ofloxacin and its chiral isomers in surface water, the influences of Fe3+ and HA content should be considered for verifying the applicability of the 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.

Erratum to: Chiral ligand-exchange high-performance liquid chromatography with copper (II)-L-phenylalanine complexes for separation of 3,4-dimethoxy-α-methylphenylalanine racemes

L-3, 4-dimethoxy-α-methylphenylalanine (L-DMMD) is an important intermediate for the synthesis of 3-hydroxy-α-methyl-L-tyrosine (L-methyldopa). This paper describes an efficient, accurate, and low-priced method of high-performance liquid chromatography (HPLC) using chiral mobile phase and conventional C18 column to separate L-DMMD from its enantiomers. The effects of ligands, copper salts, organic modifiers, pHs of mobile phase, and temperatures on the retention factors (k') and selectivity (α) were evaluated to achieve optimal separation performance. Then, thermal analysis of the optimal separation conditions was investigated as well. It was confirmed that the optimal mobile phase was composed of 20 % (v/v) methanol, 8 mM L-phenylalanine (L-Phe), and 4 mM cupric sulfate in water of pH 3.2, and the column temperature was set at 20 °C. Baseline separation of two enantiomers could be obtained through the conventional C18 column with a resolution (R) of 3.18 in less than 18 min. Thermodynamic data (∆∆H and ∆∆S) obtained by Van't Hoff plots revealed the chiral separation was an enthalpy-controlled process. To the best of our knowledge, this is the first report regarding the enantioseparation of DMMD by chiral ligand-exchange HPLC.

Chiral ligand-exchange resolution of underivatized amino acids on a dynamically modified stationary phase for RP-HPTLC.

The synthesis of Spi(τ-dec), derived from the selective alkylation of L-spinacine (4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid) at the τ-nitrogen of its heteroaromatic ring, with a linear hydrocarbon chain of 10 carbon atoms, is described here for the first time. Spi(τ-dec) was successfully employed in the past to prepare home-made chiral columns for chiral ligand-exchange high-performance liquid chromatography. In the present article a new method is described, using Spi(τ-dec) as a chiral selector in high-performance thin-layer chromatography (HPTLC): commercial hydrophobic plates were first coated with Spi(τ-dec) and then treated with copper sulfate. The performance of this new chiral stationary phase was tested against racemic mixtures of aromatic amino acids, after appropriate optimization of both the conditions of preparation of the plates and the mobile phase composition. The enantioselectivity values obtained for the studied compounds were higher than those reported in the literature for similar systems. The method employed here for the preparation of chiral HPTLC plates proved practical, efficient, and inexpensive.

Chiral separation and determination of ofloxacin enantiomers by ionic liquid-assisted ligand-exchange chromatography

A simple and accurate method for the separation and determination of ofloxacin enantiomers was developed by ionic liquid-assisted ligand-exchange high performance liquid chromatography. Both achiral and chiral ionic liquids were tested for their efficiency of ofloxacin enantiomeric separation. The effects of ligands, concentration of metal ion, organic modifier, pH of the mobile phase, and temperature were also investigated and evaluated. Optimal conditions were obtained on a conventional C(18) column, where the mobile phase consisted of methanol/water (20 : 80, v/v) (containing 4.0 mmol L(-1) amino acid ionic liquid and 3.0 mmol L(-1) copper sulfate) at a flow rate of 0.5 mL min(-1). Under this condition, the ofloxacin enantiomers could be baseline separated within 14 minutes; the proposed method was used to analyze different commercial ofloxacin medicines.

Rapid chiral separation and impurity determination of levofloxacin by ligand-exchange chromatography

A sensitive, simple, and accurate method for determination of levofloxacin and its ( R)-enantiomer was developed to determine the chiral impurity of levofloxacin in Cravit Tablets material by ligand-exchange high performance liquid chromatography. The effects of different kinds of ligands, concentration of ligands in mobile phase, organic modifier, pH of mobile phase, and temperature on enantioseparation were investigated and evaluated. Chiral separation was performed on a conventional C 18 column, where the mobile phase consisted of a methanol–water solution (containing10 mmol L −1 l-leucine and 5 mmol L −1 copper sulfate) (88:12, v/v) and its flow-rate was set at 1.0 mL min −1. The conventional C 18 column offers baseline separation of two enantiomers with a resolution of 2.4 in less than 20 min. Thermodynamic data (ΔΔ H and ΔΔ S) obtained by Van’t Hoff plots revealed the chiral separation is an enthalpy-controlled process. The standard curves showed excellent linearity over the concentration range from 0.5 to 400 mg L −1 for levofloxacin and its ( R)-enantiomer. The linear correlation equations are: y = 1.33 × 10 5 x + 6297 ( r = 0.9991) and y = 1.34 × 10 5 x + 3565 ( r = 0.9997), respectively. The relative standard deviation (RSD) of the method was below 2.3% ( n = 3).

Preparation and Application of Isoquinolinecarboxylic Acid Derivative as Chiral Stationary Phase for Ligand Exchange Chromatography

AbstractA novel chiral stationary phase (C16-(S)-THIQCA) for ligand exchange high-performance liquid chromatography was prepared by synthesizing 2-(2-hydroxyl) hexadecyl-(S)-1,2,3,4-tetrahydro-3-isoquinolinecarboxylic acid as a chiral selector and coating it on ODS-bonded stationary phase. The enantiomeric resolutions of 16 D,L-amino acids were achieved on C16-(S)-THIQCA by using cupric acetate aqueous solution as mobile phase, column temperature 25°C, and detection at UV 254 nm. The enantioselectivities α of the D,L-amino acids separated were found to be in the range from 1.09 to 1.67 with the resolution Rs ranging from 1.0 to 4.8. The elution order of D-isomer before L-isomer was observed for all D,L-amino acids used. The effects of chromatographic conditions including flow rate, column temperature, pH, and the concentration of Cu2+ and NH4+ on the resolutions of D,L-amino acid enantiomers were discussed.

Analysis of the enzymatic racemization of d-aspartic acid to l-aspartic acid by the on-line coupling of a solid-phase extraction column and a ligand-exchange high-performance liquid chromatography column

d-Aspartic acid can be enzymatically biotransformed with d-amino acid oxidase and aminotransferase to l-aspartic acid. The reaction was surveyed at three temperatures and a period of 3 days, however, l-aspartic acid can be produced only at the reaction temperature 90°C. However, the separation of d-aspartic acid and l-aspartic acid by ligand-exchange chromatography showed matrix interference. Therefore, the column-switching technique by coupling a solid-phase extraction (SPE) column to the analytical ligand-exchange HPLC column was used to eliminate the matrix effect. The pretreatment of reaction samples with the SPE column was considered as a combination of size-exclusion chromatography and ion-pair chromatography. The ion-pair reagent was 0.005 M sodium 1-octanesulfonate aqueous solution adjusted to pH 2.2. Part of the first eluted peak from the SPE column was then switched through the ligand-exchange column and analyzed with a 0.25 m M Cu 2+ aqueous mobile phase of pH 3.6. The quantitative analysis of d- and l-aspartic acids was performed by the standard addition method. Overall, the separation and analysis of d- and l-aspartic acids in the enzymic solution was convenient, fast, and successful with the developed on-line LC–LC column-coupling and column-switching system.

Enantiomeric determination of L- and D-lactic acid in human cerebrospinal fluid by chiral ligand exchange high-performance liquid chromatography.

Enantiomeric determination of L- and D-lactate in human cerebrospinal fluid (CSF) was achieved by HPLC on a chiral stationary phase with UV detection. Samples were submitted to a solid-phase extraction procedure using Oasis HLB Plus Extraction Cartridge and L- and D-lactate in the extract were separated by Shodex ORpac CRX-453 B column, a ligand exchange column for chiral separation, using a mobile phase containing copper (II) ion. L- and D-lactate were determined in 25 min. Intra-assay precision in CSF was 4.98% (mean 1.85 mmol/L) for L-lactate and 10.1% (mean 4.96 micromol/L) for D-lactate (n = 5). Detection limits were between 1.0 (L-lactate) and 1.5 (D-lactate) pmol. The mean values (n = 3) of analytical recovery for L- and D-lactate were 95% and 107%, respectively. The mean +/- SD of concentrations of L- and D-lactate in CSF (n = 20) were 1.52 +/- 0.54 mmol/L and 10.98 +/- 5.15 micromol/L, respectively.

On-line electrochemical detection of carbohydrates coupled with the periodate oxidation

A new concept of an amplified electrochemical detection of carbohydrates is proposed, where carbohydrates are oxidized by periodate ion (IO 4 −) in acidic solutions to yield iodate ion (IO 3 −) which would be electrochemically reduced into iodide ion (I −) under suitable conditions. This scenario allows highly sensitive detection of carbohydrates, for example, as 30 electrons per aldohexose molecule. Our cyclic voltammetric study revealed that IO 3 − is reduced at much lower overpotentials than IO 4 − at gold, platinum and carbon-based electrodes despite the fact that the standard redox potential of the IO 3 −/I − couple is more negative than that of the IO 4 −/I − couple. In the electrochemical reduction of IO 3 −, I − is considered to function as a mediator. Stable flow-through detection of IO 3 − in the presence of IO 4 − was realized at glassy carbon electrodes. This method was coupled with the IO 4 − oxidation of carbohydrates and the experimental conditions were partially optimized on a flow injection system. The IO 4 − oxidation-coupled electrochemical detection of carbohydrates was applied to ligand-exchange high-performance liquid chromatography in a post-column mode. Sub-nanomole order of carbohydrates were successfully detected on this system.

Enantiomeric Separation of Non-Protein Amino Acids by Chiral Ligand-Exchange High-Performance Liquid Chromatography

The enantiomeric separation by high-performance liquid chromatography of underivatized non-protein amino acids was investigated by using a column packed with octadecylsilanized silica coated with N,S-dioctyl-D-penicillamine as a chiral ligand-exchange phase (Sumichiral OA-5000). Excellent to good separations of enantiomers were achieved with a variety of nonprotein amino acids carrying aliphatic or aromatic side-chains by optimizing the amount (0-20%, v/v) of the organic component (2-propanol) and the concentration (1-5 mM) of the complexing metal ion (Cu2+) in the hydro-organic eluent.

Direct enantiomeric separation of platelet-activating factor receptor antagonist SM-10661 by ligand-exchange high-performance liquid chromatography with a copper (II) N,S-dioctyl-D-penicillamine complex.

The enantiomeric separation of SM-10661, a platelet activating factor receptor antagonist, was investigated by HPLC using ligand-exchange chiral stationary phases. The stereoisomers of SM-10661 could all be separated by ligand-exchange HPLC with a Cu(II) N,S-dioctyl-D-penicillamine complex (Sumichiral OA-5000). The mechanism for the resolution includes the involvement of hydrophobic interactions between SM-10661 and Cu(II) D-penicillamine.

Enantiomeric separation and determination of antiparkinsonian drugs by reversed-phase ligand-exchange high-performance liquid chromatography

A simple and rapid high-performance liquid Chromatographic method for the separation and determination of enantiomers of levodopa and carbidopa using a LiChrosper C 18 column with aqueous copper- l-phenylalanine as mobile phase was developed. The separation between d- and l-enantiomers of levodopa and carbidopa was fairly good with separation factors of 1.63 and 2.38, respectively. The method was validated using synthetic mixtures and used for quality assurance of commercial formulations.

Ligand-exchange high-performance liquid chromatography of fluorine-containing phenylglycine and phenylalanine

The relationships between the number of fluorine atoms, their position in the aromatic ring of fluorine-containing phenylglycine and phenylalanine and the selectivity of the separation of enantiomers on Chiral ProCu, Chiral ValCu and Chiral-1 (hydroxyproline) columns were studied. Optimum conditions for the separation of enantiomers were established.

Chiral separation of unmodified amino acids by ligand-exchange high-performance liquid chromatography using copper(II) complexes of l-amino acid amides as additives to the eluent

Copper(II) complexes of l-amino acid amides added to the eluent in reversed-phase high-performance liquid chromatography are able to perform chiral discrimination of unmodified amino acids with high enantioselectivity. The mechanism is consistent with a ligand exchange between the binary initial copper(II) complex and the enantiomers. Evidence is provided that the exchange of the ligand is actually occurring during the chromatographic separation. The system involves a series of equilibria of exchange in the aqueous solution, in the stationary phase and between the two phases. Enantioselectivity is essentially due to the adsorption of the diastereomeric ternary species on the column, whereas the relative stabilities of the mixed complexes in the mobile phase seem to be negligible with respect to the overall discrimination process. The structural features of the initial copper complexes greatly affect the stereoselectivity of the process. The chromatographic parameters (pH, selector concentration, eluent polarity, ionic strength) are examined.

Direct separation of enatiomers by high-performance liquid chromatography on a new chiral ligand-exchange phase

Two novel chiral ligand-exchange phases for high-performance liquid chromatography, N,S-dioctyl- d-penicillamine (I) and N,S-dioctyl-N-methyl-( d-penicillamine (II) were prepared. The direct separation of more than twenty amino acid enantiomers was achieved using octadecylsilanized silica coated with these phases and water or hydro-organic eluents containing copper(II) ion as a mobile phase. Various enantiomers including amino acid derivatives, hydroxy acids and amino alcohols were also resolved directly. Generally, phase I showed higher enantioselectivity than phase II, which, however, was very efficient for the separation of some long-retained racemic compounds such as tryptophan and madelic acid. These phases are very promising for the direct separation of a variety of enantiomers by ligand-exchange high-performance liquid chromatography.

Preparative separation and analysis of the enantiomers of [ 3H]Abbott-69992, an HIV anti-infective nucleoside, by ligand-exchange high-performance liquid chromatography

Several chiral stationary phases (CSPs) were examined to separate the enantiomers of A-69992, a chiral HIV anti-infective nucleoside. The only CSP found to be effective was Nucleosil Chiral-1, a ligand-exchange CSP, which was used to prepare microgram amounts of the enantiomers of high optical purity. This appears to be the first separation of the enantiomers of a nucleoside by chiral high-performance liquid chromatography.

Ion-Pair, anion-exchange and ligand-exchange high-performance liquid chromatography of tenuazonic acid and 3-acetyl 5-substituted pyrrolidine-2,4-diones

The ion-pair, ligand-exchange and anion-exchange chromatography of the fungal metabolic tenuazonic acid (TA) and its related 3-acetyl 5-substituted pyrrolidine-2,4-diones were studied. Ion-pair chromatography was performed on a C 18 column with a mobile phase composed of cetrimide, phosphate buffer in water-methanol and a metal complexant (ethylenediamine) to improve the peak sharpness. Addition of the same metal complexant to the mobile phase of the anion-exchange chromatographic system also improved its efficiency. TA and its 5-substituted analogues derived from valine and leucine were separated with the ion-pair and anion-exchange chromatographic systems. With ligand-exchange chromatography, TA could only be separated from its valine analogue. These chromatographic systems were used for the detection of TA in the culture filtrates of the fungus Pyricularia oryzae and in infected rice leaves. Deproteinated culture filtrates could be rapidly analysed for their TA content by anion-exchange chromatography. However, this system was not suitable for the detection of TA in the infected rice leaf as interfering compounds were coeluted with TA. Ion-pair and ligand-exchange chromatographic systems allowed the efficient quantification of TA in infected leaves.

A Novel Chiral Stationary Phase for Optical Resolution of Amino Acids and Their Derivatives by Ligand-Exchange High-Performance Liquid Chromatography

Abstract A novel chiral stationary phase, [(1R,2S)-2-hydroxy-1-methyl-2-phenylethylamino]acetic acid, was prepared from (−)-norephedrine, and its sodium salt was bound to silica gel pretreated with [3-(glycidyloxy)propyl]trimethoxysilane. The chiral stationary phase was found to be effective for the optical resolution of amino acids and their derivatives by ligand-exchange high-performance liquid chromatography using aq copper(II) sulfate solution as an eluent.

Enantiomeric resolution of N-methyl-α-amino acids and α-alkyl-α-amino acids by ligand-exchange chromatography

The enantiomeric resolution of N-methyl-DL-α-amino acids (NMe-AA) and DL-α-alkyl-α-amino acids (AAA) was achieved by ligand-exchange (LE) high performance liquid chromatography (HPLC) using silica bonded L-amino acids as the chiral selector (Chiral ProCu, Chiral HyproCu, Chiral ValCu, Nucleosil Chiral-1). Using aqueous solutions of copper sulfate or acetate adjusted to pH 4–6 and the addition of acetonitrile (10–20%) or methanol (10–40%), baseline resolution occurred in many cases. By comparison with enantiomers of known optical configuration, the elution order of a number of NMe-AA and AAA could be determined and on-line polarimeter detection enabled the assignment of the optical rotation, elution order and configuration of AAA enantiomers. Using a preparative column (300mm × 20mm i.d.) packed with Chiral ProCu, a baseline resolution of 60mg DL-α-methylphenylalanine was achieved. Further, LE-TLC, using a chiral stationary phase (“Chiralplate”), enabled the chiral separation of very hydrophobic AAA, characterized by increasingly large alkyl side chains,viz. the homologous series α-ethylalanine to α-nonylalanine and α-propyl-α-amino-n-butyric acid to α-hexyl-α-amino-n-butyric acid.