Liquid chromatography determination of citalopram enantiomers using beta-cyclodextrin as a chiral mobile phase additive

A reliable and specific method for the determination of citalopram enantiomers was developed and validated. Chromatographic resolution of citalopram enantiomers was made on a Shim-pack (5 microm particle size) cyanopropyl column with beta-cyclodextrin (beta-CD) as an effective chiral mobile phase additive. The composition of the mobile phase was (90 + 10, v/v) aqueous 0.1% triethylammonium acetate buffer, pH 4.0 (adjusted with acetic acid), and acetonitrile, containing 12 mM beta-CD. The flow rate was 0.8 mL/min with ultraviolet detection at 240 nm. The effects of the mobile phase composition, concentration of beta-CD, and pH of the triethylammonium acetate buffer on peak shape and resolution of the enantiomers were investigated. The calibration graphs were linear (r = 0.9999, n = 8) in the range of 1-40 microg/mL for S(+) citalopram and R-(-) citalopram. The limit of detection values were 5.51 x 10(-3) and 4.35 x 10(-3) pg/mL, while the limit of quantification values were found to be 1.84 x 10(-2) and 1.45 x 10(-2) microg/mL for S-(+) citalopram and R-(-) citalopram, respectively.     

Screening for amino acid disorders by thin-layer chromatography of the dansyl amino acids

Summary A procedure is described for the screening of disorders of amino acid metabolism or tranpsort. The amino acids and other reactive constituents present in a small volume of deproteinized plasma or urine are derivatized with dansyl chloride. Desalting or concentrating of urine is not required. The fluorescent derivatives are separated by two-dimensional thin-layer chromatography and visualized by ultraviolet radiation. The time of development is less than 1 hr. The derivatives of thirty-seven amino acids and related constituents found in physiological fluids have been mapped in the system. A standard, reflective of normal plasma, is used to aid in identification of those derivatives generally observed on plasma and urine chromatograms and in detection of aminoacidemias. Aminoacidurias are detected by observing derivatives that in relation to others are present in greater than normal amounts.

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Analytischer Nachweis von Störungen des Aminosäurestoffwechsels durch Dünnschicht-Chromatographie der Dansylverbindungen

Zusammenfassung Dansylderivate von Aminosäuren und anderen reaktionsfähigen Stoffen in kleinen Volumina enteiweißten Plasmas oder Harns wurden hergestellt. Entsalzung oder Einengung des Harns ist nicht notwendig. Die fluoreszierenden Derivate werden durch zweidimensionale Dünnschichtchromatographie getrennt und im UV nachgewiesen. Die dazu erforderliche Zeit ist kürzer als 1 Stunde. Die Derivate von 37 Aminosäuren und verwandten Verbindungen in physiologischen Flüssigkeiten wurden erfaßt. In üblicherweise zur Beobachtung gelangenden Plasma- bzw. Harnchromatogrammen feststellbare Aminosäuren dienen zum Vergleich mit abnormalen Ergebnissen.

     

Reversed phase planar chromatography of dansyl DL amino acids with bovine serum albumin in the mobile phase

Summary The chromatographic behavior of twelve dansyl DL amino acids, one D isomer and eleven L isomers on RP₁₈W/UV₂₅₄, RP₁₈W/F_254s, and Sil C₁₈–50 UV₂₅₄ plates developed with aqueous—organic solutions containing bovine serum albumin (BSA) as chiral complexing agent has been extensively investigated. Enantiomeric resolution is highly dependent on mobile phase pH and ionic strength, and on the concentration of both BSA and organic modifier. All the racemates have been resolved within a development time of 1 h 30 min. The selectivity factors () for the dansyl amino acids have been compared with those from planar chromatography for the corresponding DNP, DNPy, and Fmoc amino acids, and with those of the same dansyl derivatives on a column prepared from BSA bound to silica gel.     

Chromatographic behavior of ion pair enantiomers of dansyl leucine cyclohexylammonium salt on a beta-cyclodextrin stationary phase and the effect of a competitive-binding mobile phase additive

The separation of dansyl leucine enantiomers on a beta-cyclodextrin stationary phase is significantly complicated by the association of the amino acid with its cyclohexylammonium counter ion, in a mobile phase of 80:20 (v/v) methanol-water. This produces very unusual chromatography, with two partially superimposed peaks observed for each enantiomer at lower column temperatures. The peak shape is attributed to the irreversible, oncolumn conversion of the ion pair (I) to the free, protonated (neutral) dansyl amino acid (II+H). Increasing the ionic strength of the mobile phase greatly improves the chromatography by transforming the solute species to enantiomers of II (the anionic, free amino acid). Van't Hoff plots are constructed for both species I and II (under different mobile phase conditions) to provide thermodynamic insight into the major enantioselective driving forces of separation. The chiral discrimination of the stationary phase is found to be primarily enthalpically driven for both solutes. Finally, 1-adamantanecarboxylic acid (ACA) is investigated as a solute-competitive mobile phase additive to intentionally block the hydrophobic cyclodextrin cavities on the stationary phase. By varying the concentration of ACA additive in the mobile phase, control over the retention and chiral recognition of the stationary phase is demonstrated.     

Determination of D-alanine and D-glutamic acid in biological samples by coupled-column chromatography using beta-cyclodextrin as mobile phase additive.

A high-performance liquid chromatographic assay for monitoring traces of D-alanine and D-glutamic acid in peptide and protein hydrolysates is presented, which uses a two-column set-up with column switching. The main advantage of the proposed method is the higher reliability of the results, compared with the analysis by derivatization and single-column chromatography of the diastereomers. A non-chiral alkyl-silica reversed-phase column is combined with a second column, in which beta-cyclodextrin is used as a chiral mobile phase additive. The amino acids of the hydrolysate are dansylated, and the amino acid of interest is separated from the others on the first column and transferred to the second column where chiral resolution is performed. The transfer volume of ca. 200 microliters is small enough not to cause any peak distortion or dilation in the second column.     

Ion-pair reversed-phase thin-layer chromatography of basic drugs using sulphonic acids

The use of sulphonic acid ion-pair reagents in the thin-layer chromatography of four basic drugs (all secondary amines) on C18-bonded silica gel, paraffin coated silica gel and silica gel itself has been investigated. Effects of the ion-pair reagents were only obtained on C18-bonded silica gel, and only then when the reagents were pre-coated onto the stationary phase. In general the largest reductions in the RF values of the test compounds occurred when sodium dodecylsulphate was coated onto the plates.     

Enantiomeric resolution of dansyl phenylalanine and analogs by microcolumn liquid chromatography with γ-cyclodextrin as mobile phase additive

Dansyl derivatives of racemic phenylalanine and its analogs have been resolved by microcolumn liquid chromatography with γ-cyclodextrin as mobile phase additive. The enantioselectivity of the system was influenced by the concentrations of both γ-cyclodextrin and acetonitrile in the mobile phase. Enantiomers of phenylalanine, phenylglycine, and tyrosine were resolved by isocratic elution in a single chromatographic run.     

以L-酒石酸正己酯-硼酸配合物作为反相液相色谱手性流动相添加剂拆分5种β-受体阻滞剂

本文以L-酒石酸正己酯-硼酸配合物为手性流动相添加剂,建立了普萘洛尔、艾司洛尔、美托洛尔、比索洛尔、索他洛尔和阿替洛尔六种β-受体阻滞剂的反相高效液相色谱手性分离方法。对影响对映体分离的主要因素：L-酒石酸正己酯、硼酸浓度,缓冲溶液种类、浓度、pH值和有机改性剂-甲醇含量等进行了详细考察。最佳色谱条件为：Venusil MP-C18色谱柱（4.6 mm × 250 mm,5 μm）,流动相为15 mmol/L乙酸铵-甲醇（体积比为20: 80或30: 70,含60 mmol/L硼酸,70 mmol/L L-酒石酸正己酯,醋酸调节pH值6.00）,检测波长214 nm。在最佳分离条件下,五对对映体（普萘洛尔、艾司洛尔、美托洛尔、比索洛尔、索他洛尔）可以分别获得基线分离。     

Stereoselective determination of p-hydroxyphenyl-phenylhydantoin enantiomers in rat liver microsomal incubates by reversed-phase high-performance liquid chromatography using beta-cyclodextrin as chiral mobile phase additives

An analytical method was developed for determination of p-hydroxyphenylphenylhydantoin enantiomers in rat liver microsome by using reversed-phase high-performance liquid chromatography. A 50 mm C(8) column was used as the analytical column. The mobile phase was made up of 8.8 mmol/L beta-cyclodextrin, 0.25 mol/L urea and 0.05 mol/L ammonium acetate in water. The assay was linear from 2.05 to 410.0 micromol/L for each enantiomer. The limits of detection and of quantitation for the method were 0.90 and 2.05 micromol/L for each enantiomer, respectively. The analytical method afforded average recoveries of 93.59 +/- 2.75% and 94.72 +/- 1.78% for S- and R-p-hydroxyphenylphenylhydantoin, respectively. The method allowed study of the in vitro glucuronidation of p-hydroxyphenylphenylhydantoin in rat liver microsomal incubates. The stereoselectivity of p-hydroxyphenylphenylhydantoin phase II metabolism was observed.     

Dimethylformamide as a mobile phase component in reversed-phase liquid column chromatography on octadecylsilica

Summary N,N-Dimethylformamide selectively accelerates the elution of weakly basic compounds and reduces their tailing in reversed-phase liquid chromatography with aqueous organic mobile phases. This effect is demonstrated with two types of octadecylsilica packings for representative solutes, covering aromatic amines and N-heterocyclic compounds such as pyridine, pyrimidine and purine derivatives.     

High-speed assay of amino acids using reversed-phase liquid chromatography

A procedure is described by which 20 commonly occurring amino acids may be separated by reversed-phase liquid chromatography at room temperature in a total time, including derivatisation with o-phthalaldehyde/2-mercaptoethanol reagent, of approximately 15 minutes. Further, an increase in column temperature enables increased resolution of certain amino acids to be obtained with small but acceptable losses in fluorescence intensity. The only major drawback to the method is that the derivatisation reagent does not react with proline or hydroxyproline.     

Determination of mephenytoin stereoselective oxidative metabolism in urine by chiral liquid chromatography employing beta-cyclodextrin as a mobile phase additive.

A direct high-performance liquid chromatographic assay was developed for the separation and determination of S- and R-mephenytion enantiomers in human urine. Separation was achieved on a Supelcosil LC-8 column with methanol-0.1 M acetate buffer as the mobile phase and beta-cyclodextrin as a mobile phase additive. The urinary S/R enantiomeric ratio of mephenytoin was measured in a 32-h urine sample (as phenotypic traits) for determination of the ability of normal subjects to hydroxylate racemice mephenytoin after oral administration of the drug.     

Selectivity of dansyl amino acids in micellar liquid chromatography with an ion-exchange-induced stationary phase

Summary The retention behavior of dansyl amino acids in micellar liquid chromatography has been examined by using ionexchange-induced stationary phases. Several parameters affected the retention of the analytes, including the type and concentration of micellar agent and modifier ion and the concentration of acetonitrile in the mobile phase. The order of elution of dansyl amino acids obtained with the micellar mobile phase was very different from that observed in conventional reversed-phase liquid chromatography. Fluorescence intensities of some dansyl amino acids were enhanced by the micellar mobile phase.     

Possibility of calculating the Rm values in thin-layer chromatography using a ternary mobile phase

Summary On the basis of an equation derived it is possible to establish theoretically the R_M values for a ternary mobile phase. The R_M values of phenol and its derivatives, naphthalene and its derivatives and some polycyclic aromatic hydrocarbons were measured. A good agreement has been found between the calculated and the experimentally obtained R_M values. Analysis of the results showed some regularities of certain values obtained in binary systems when related to the ternary mobile phase.     

Concentration of sodium ion as the determining factor for the association of dansyl amino acids with the teicoplanin molecule in reversed-phase liquid chromatography

The mechanism of the binding of D,L dansyl amino acids to teicoplanin was investigated. Na+ was used as an indicator of the interactions between the solutes and teicoplanin. The number (n) of sodium ions, Na+, excluded from the solute-teicoplanin interface when analyte transfer occurred was determined. A thermodynamic study and enthalpy-entropy compensation were performed to further explore the interaction mechanism. From these results, it was shown that teicoplanin was balanced between 2 conformational states characterized by distinct enantioselective properties. This approach indicates that liquid chromatography (LC) is a useful tool to extract physicochemical and molecular information from retention data. Thus, LC can be used as a complementary technique with the conventional techniques of molecular interaction analysis.     

Cyclodextrins as a chiral mobile phase additive in nano-liquid chromatography: comparison of reversed-phase silica monolithic and particulate capillary columns

Enantioseparations of racemic nonsteroidal anti-inflammatory drugs (naproxen, ibuprofen, ketoprofen, flurbiprofen, suprofen, indoprofen, cicloprofen, and carprofen) were performed by nano-liquid chromatography, employing achiral capillary columns and heptakis(2,3,6-tri-O-methyl)-β-cyclodextrin (TM-β-CD) or hydroxylpropyl-β-cyclodextrin (HP-β-CD) as a chiral mobile phase additive (CMPA). Working under the same experimental conditions (in terms of mobile phase and linear velocity), the performance of a RP-C18 monolithic column was compared with that of a RP-C18 packed column of the same dimensions (100 μm i.d. × 10 cm). Utilizing a mobile phase composed of 30% ACN (v/v) buffered with 50 mM sodium acetate at pH 3, and containing 30 mM TM-β-CD, the monolithic column provided faster analysis but lower resolution than the packed column. This behavior was ascribed to the high permeability of the monolithic column, as well as to its minor selectivity. HP-β-CD was chosen as an alternative to TM-β-CD. Employing the monolithic column, the effects of different parameters such as HP-β-CD concentration, mobile phase composition, and pH on the retention factor and the chiral resolution of the analytes were studied. For the most of the analytes, enantioresolution (which ranged from R _s = 1.80 for naproxen to R _s = 0.86 for flurbiprofen) was obtained with a mobile phase consisting of sodium acetate buffer (25 mM, pH 3), 10% MeOH, and 15 mM HP-β-CD. When the same experimental conditions were used with the packed column, no compound eluted within 1 h. Upon increasing the percentage of organic modifier to favor analyte elution, only suprofen eluted within 30 min, with an R _s value of 1.14 (20% MeOH). Replacing MeOH with ACN resulted in a loss of enantioresolution, except for naproxen (R _s = 0.89).     

Influence of inorganic mobile phase additives on the retention and separation efficiency of selected amino acids in thin-layer chromatography on cellulose layers

Selected amino acid standards are investigated on cellulose layers using organic-aqueous eluent systems modified with neutral and chaotropic salts: chlorides, iodides, nitrates, thiocyanates, perchlorates, and hexafluorophosphates at low concentrations ranging from 10 up to 80mM in whole mobile phase. The effect of salts used as the mobile phase modifier is analyzed by the comparison of densitograms, peak symmetry coefficient, and theoretical plate number. The efficiency of chromatographic systems modified with inorganic salts additives depends primarily on the kind of salt and organic solvent in the mobile phase. The best efficiency is obtained through the addition of ammonium thiocyanate to the mobile phase containing acetonitrile as an organic modifier.