Reversed-phase Ion-pair System Research Articles

Separation and identification of the regioisomers of verdoheme by reversed-phase ion-pair high-performance liquid chromatography, and characterization of their complexes with heme oxygenase

We report an HPLC method for separating the four regioisomers of verdoheme formed in the coupled oxidation of hemin with oxygen and ascorbate in aqueous pyridine. The reversed-phase ion-pair system uses hexafluoroacetone and pyridine as ion-pair agents. The regiochemistry of the separated isomers was established both by HPLC of the corresponding biliverdin IX derivatives and by 1H NMR of each isomer. Optical spectra of the pyridine verdohemochrome isomers were similar to each other, but showed differences in the absorption maxima in the red region, which appear at 680, 663, 648 and 660 nm for the α, β, γ, and δ-isomers, respectively. Each of the four isomers was incorporated anaerobically into heme oxygenase-1, yielding the corresponding verdoheme–enzyme complex. The ferrous forms had absorption maxima at 690, 667, 655, and 663 nm, and their CO-bound forms had maxima at 638, 624, 616, and 626 nm for α, β, γ, and δ-isomer, respectively. Addition of ferricyanide to the α-verdoheme–heme oxygenase complex brought about a ferric low-spin heme-like signal, which is identical with the ferric α-verdoheme complexed with the heme oxygenase that was observed in the heme oxygenase reaction.

High–Performance Liquid Chromatographic Analysis of Chlorhexidine in Saliva after Mouthrinsing

A high–performance liquid chromatographic method was developed to quantify chlorhexidine in human saliva. After addition of an internal standard and acidicdeproteinization, saliva samples were chromatographed by the reversed–phase ion–pair system using pentanesulfonate as a counterion and the eluates were detected by a diode array detector. Under optimal conditions, the baseline separation of analytes was achieved within 4.5min without interference from components in saliva matrix. The method showed high selectivity to salivary chlorhexidine, quantitative range (50.0ng/ml–50.0μg/ml), recovery (96.0–97.8%) and analytical precision (intra– and interassay CVs within 0.41%), permitting the effective application to both saliva and aqueous solutions. Chlorhexidine was found in saliva at microgram per milliliter levels for at least 8 h after mouthrinsing with 10 ml of an aqueous solution of chlorhexidine (0.1 or 1.0 mg/ml) for 1 min. The concentrations of salivary chlorhexidine were reduced by ingesting the acidic beverage and food, but not by ingesting the neutral beverage. The adsorption experiments in artificial saliva revealed that chlorhexidine was significantly adsorbed to saliva–coated hydroxyapatite, buccal epithelial cells and mucin. The proposed method will be a useful tool to study salivary chlorhexidine after oral application and its retention in the oral cavity.

Assay for the (R)- and (S)-enantiomers of salsolinols in biological samples and foods with ion-pair high-performance liquid chromatography using beta-cyclodextrin as a chiral mobile phase additive.

A chromatographic procedure was devised for the quantitative determination of the enantiomers of salsolinol and N-methylsalsolinol, which are biologically important alkaloids. The enantiomers of salsolinol and N-methylsalsolinol were completely separated using beta-cyclodextrin in a reversed-phase ion-pair system. The HPLC method was sensitive enough to detect the isoquinolines at a concentration less than 0.1 pmol per injection. The presence of (R)- and (S)-salsolinol was confirmed in fermented foods and beverages, while N-methylsalsolinol was not detected. On the other hand, the (R)-enantiomers of both salsolinol and N-methylsalsolinol were found to predominate in the human brain.

Sensitive and selective bioanalytical assay for the determination of dobutamine in rat plasma samples

Dobutamine is one of the synthetic catecholamines acting directly onβ1-receptors. For the analysis of dobutamine in rat plasma samples, a selective and sensitive liquid chromatographic method is described. After a simple liquid-liquid extraction, separation of the analyte was performed using a reversed-phase ion-pair system with an octyl modified silica column. The solute was detected by fluorescence detection, applying an excitation wavelength of 285nm and an emission wavelength of 313nm. The (im)possibilities of the application of the normally used assays for the isolation, concentration and quantitation of catecholamines are discussed. By the addition of a minimum amount of modifier to the mobile phase, the selectivity of the system was increased significantly. With this method the detection limit is 9ng/ml in 0.2ml plasma samples. The application of the method is shown in rat plasma samples by measuring the concentration-time curves to establish plasma level-effect relationships for this drug.

Reversed-phase ion-pair systems to predict partition coefficients of β-carbolines by HPLC and TLC

A group of 17 β-carbolines was studied in HPLC and TLC systems in order to predict their partition coefficients (log P values). On account of the basic or acid character of some of these compounds, an ion pairing system gave the best results. Both HPLC and TLC data were comparable for log P prediction but severe pH conditions required the use of TLC plates. Retention data are quantitatively related to lipophilicity (expressed as the Hansch constant) and polarity (as the inductive constant) of the solute molecule.

Selected aspects of the development of methods for the analysis of drugs by high performance liquid chromatography.

The successful analysis of drugs in biological fluids and other matrices by reversed phase high performance liquid chromatography (h.p.l.c.) relies upon the optimization of the chromatographic separation, the sample preparation and the post-column detection. Recent advances in the detection and derivatisation of exogenous substances are described elsewhere in this volume (Sternson 1986). This paper deals with selected aspects of the optimization of the chromatographic separation, concentrating on the theoretical basis and practical application of mobile phase selectivity in reversed phase ion-pair systems. In addition, recent advances in sample preparation techniques which permit the direct injection of biological fluids are discussed.

Reversed-phase ion-pair systems for the prediction of n-octanol-water partition coefficients of basic compounds by high-performance liquid chromatography

The correlation between the retention of basic solutes in a reversed-phase ion-pair system, in which sodium dodecylsulphate is the pairing-ion, and their n-octanol-water partition coefficients (log P) has been studied. The study comprises the influence on the correlation of the type hydrophobic matrix (phenyl- and octadecylsilica) and of the pH and salt concentration in the mobile phase. The influence of the ion-pairing additives on the prediction of log P of neutral compounds from their retention has also been studied. The results show that the correlation between log P and log k′ for basic compounds is significantly better on phenylsilica than on octadecylsilica. The error in the prediction of log P, ranging from 1 to 7, of basic compounds from their retentions in the ion-pair system on phenylsilica was found to be ± 0.1 log units at a 95% confidence limit. The correlation of log P values of neutral compounds with their k′ values in the ion-pair system is not significantly worse than that found with a normal reversed-phse system. Moreover, it seems that the slopes of the linear regression of log P and log k′ of neutral and basic compounds in the selected phases system are about equal. This permits the prediction of log P of neutral and basic compounds from their retention in the same phases system with an error in the prediction of ± 0.1 long units at a 95% confidence limit.

High-performance liquid chromatographic methods for the determination of ranitidine and its metabolites in biological fluids

The use of an existing reversed-phase ion-pair method for the determination of ranitidine, ranitidine-N-oxide, ranitidine-S-oxide and desmethylranitidine in the plasma of patients taking the anti-ulcer drug, ranitidine is described. The development of a ternary reversed-phase system which is more suitable for the routine determination of ranitidine and the three metabolites is reported. This system has been used to determine quantitatively ranitidine and the metabolites in urine. Studies in animals using14C-ranitidine have shown that ranitidine is also oxidatively deaminated to a 5-substituted, 2-furan carboxylic acid. A reversed-phase ion-pair system, in which cetrimide is the counter ion, has been developed for the quantitative determination of the 5-substituted, 2-furan carboxylic acid and ranitidine-N-oxide in urine and faeces from patients treated with ranitidine.

High-performance liquid chromatographic method for the determination of bamifylline and its three metabolites in human plasma

The performance of a gradient elution normal-phase system and of two isocratic elution reversed-phase ion-pair systems was investigated for the high-performance liquid chromatographic separation of bamifylline and its three metabolites. Three packings and mobile phases were tested and the best separation was achieved using a reversed-phase ion-pair system with Hypersil ODS 3 μm as stationary phase. The effects of alkyl chain length and concentration of alkyl sulfonates, salt concentration, column temperature and competing amine on separation and peak tailing are discussed. The determination of bamifylline and metabolites in human plasma using the defined optimal chromatographic conditions is reported.

Simultaneous liquid-chromatographic determination of zinc protoporphyrin IX, protoporphyrin IX, and coproporphyrin in whole blood.

We describe a method for simultaneously measuring concentrations of coproporphyrin, zinc protoporphyrin IX, and protoporphyrin IX in whole blood by liquid chromatography, with use of reversed-phase ion-pair system, fluorometric detection, and internal standardization. Each analysis requires 10 microL of whole blood and 15 min total analysis time. Analytical recovery ranged from 84 to 92%, day-to-day precision (CV) from 5 to 12%. Uroporphyrin, though not studied in detail, can also be detected by this method.

Simultaneous liquid-chromatographic determination of zinc protoporphyrin IX, protoporphyrin IX, and coproporphyrin in whole blood.

Abstract We describe a method for simultaneously measuring concentrations of coproporphyrin, zinc protoporphyrin IX, and protoporphyrin IX in whole blood by liquid chromatography, with use of reversed-phase ion-pair system, fluorometric detection, and internal standardization. Each analysis requires 10 microL of whole blood and 15 min total analysis time. Analytical recovery ranged from 84 to 92%, day-to-day precision (CV) from 5 to 12%. Uroporphyrin, though not studied in detail, can also be detected by this method.