Reversed-phase Ion-pair Liquid Chromatographic Method Research Articles

Measurement of theanine using reverse-phase ion-pair liquid chromatography with photodiode array detection

There is increasing demand for theanine as a taste-enhancing additive and as a supplement for improving and maintaining human health. We describe the development of an ion-pair reversed-phase liquid chromatographic method for the analysis of theanine in tea. Samples may be directly analysed after sonication for 10min. The mobile phase consists of an aqueous 2mM solution of pentadecafluorooctanoic acid (ion pair reagent) and acetonitrile (82:18, v/v). The theanine peak is detected using a UV detector at 200nm. The limit of detection is 5.1ng and the limit of quantitation is 15.6ng. The method is accurate with recoveries higher than 97%.

Simultaneous Ion-Pair LC Analysis of Ephedrine and Pseudoephedrine in Rat Liver Microsomal Incubations: Application to In-Vitro Metabolism

(−)-Ephedrine (ephedrine, EPH) and (+)-ephedrine (pseudoephedrine, PEPH) are metabolized by the liver, but the species of hepatocyte cytochrome P450 (CYP450) responsible is not yet clear. To investigate which subtype of CYP450 is involved in the metabolism of EPH and PEPH, a rapid and reliable reversed-phase ion-pair liquid chromatographic method for simultaneous analysis of EPH and PEPH in rat liver microsomes has been established and validated. Matrine was selected as a suitable internal standard (IS) for calibration. After liquid–liquid extraction of liver microsomal samples with methyl tert-butyl ether, EPH and PEPH were separated on a C18 reversed-phase column (200 mm × 4.6 mm, 5 μm) with methanol–0.5% sodium dodecyl sulfate–phosphoric acid–triethylamine 60:40:1.25:1 (v/v) as mobile phase at a flow rate of 1.0 mL min. Detection was by UV absorbance at 210.5 nm. For both EPH and PEPH, calibration curves were linear over the range 1.5–60.0 μg mL−1, the limit of quantification was 1.5 μg mL−1, and intra-day and inter-day variability was 73%. The validated method was successfully used to study the in-vitro metabolism of EPH and PEPH. In rat liver microsomes induced by dexamethasone, enzyme activity in the metabolism of EPH and PEPH was higher than that for metabolism of phenobarbital and β-naphthoflavone.

Reversed-phase ion-pair liquid chromatographic method for determination of reaction equilibria involving ionic species: Exemplification of the method using ligand substitution reactions of ethylenediaminetetraacetatochromium(III) ion with acetate and phosphate ions

A reversed-phase ion-pair liquid chromatographic method is presented for the determination of reaction equilibria involving ionic species of the same charge sign as reactant and product compounds. It has been demonstrated that ion-exchange chromatography or reversed-phase ion-pair chromatography is a useful tool for the determination of equilibrium constants of chemical reactions involving ionic species such as metal complexation reactions. Previous work with these methods has been based on the assumption that the limiting retention factors of the reactant and product species are constant independent of concentration of the chemical species (X) in the mobile phase, which reacts with the analyte compound. However, when all the reactant and product species are ions of the same charge sign as that of the species X, it is virtually impossible to apply these methods to the equilibrium constant determination because the retention factors of both the reactant and product species may depend on the concentration of X. In this study, an alternative approach was developed that estimates the limiting retention factors of ionic species from the dependence of the retention factor on the ionic strength of the mobile phase. Ligand substitution reactions of ethylenediaminetetraacetatochromium(III) ion with acetate and phosphate ions were used as model reactions to test this method. The equilibrium constants determined by this method are in good agreement with those obtained by a UV–visible spectrophotometric method.

Simultaneous determination of levodopa and 3- O-methyldopa in human plasma by liquid chromatography with electrochemical detection

A simple and rapid assay is described for the simultaneous analysis of levodopa ( l-DOPA) and 3- O-methyldopa (3-OMD) in human plasma samples, applying an ion-pair reversed-phase liquid chromatographic method with electrochemical detection, designed for clinical trials performed to study the effect of peripheral catechol- O-methyltransferase inhibitors on the metabolism of l-DOPA. After protein precipitation of 100 μl plasma sample aliquots with perchloric acid, the analytes are directly injected, separated within 10 min and simultaneously quantified down to 20 ng/ml by an electrochemical detector equipped with a dual-electrode system operating in redox mode eliminating effectively potential endogenous and exogenous interferences. The intra-assay precision for l-DOPA and 3-OMD was 1.34–6.54 and 3.90–5.50%, whereas the inter-assay precision was 2.09–7.69 and 4.16–9.90%, respectively. The recoveries were close to 90% for l-DOPA and almost 100% for 3-OMD. Satisfactory storage stability was achieved for up to 16 weeks at −70 °C by stabilizing plasma samples with antioxidants.

Liquid chromatographic method for separation of lincomycin from its related substances

A reversed-phase ion-pair liquid chromatographic method with UV detection at 210 nm is described for the separation of lincomycin from 7-epilincomycin and lincomycin B. The method utilizes a base-deactivated Supelcosil LC-ABZ, C 12–18, 5 μm, 250×4.6 mm I.D. column maintained at 45°C. The mobile phase consists of 2.25% (v/v) acetonitrile, 5% (v/v) phosphate buffer (2.72%, m/v KH 2PO 4 adjusted to pH 5.0 with 3.48%, m/v K 2HPO 4), 0.067% (v/v) methanesulfonic acid and water to 100%. The flow-rate is 1.0 ml/min. The separation of lincomycin from 7-epilincomycin is only possible on base-deactivated (BDS) columns. Other octadecylsilyl columns examined showed insufficient selectivity. The method was also tested on other BDS columns (Spherisorb S5-ODS-B, 5 μm, Hypersil BDS, C 18, 5 μm and Supelcosil LC-ABZ, C 18, 5 μm, all 250×4.6 mm I.D.) and showed good robustness. Robustness was further evaluated by performing a full-factorial design experiment. The method showed good selectivity, repeatability, linearity and sensitivity. It is also suitable for analysis of lincomycin formulations.

Liquid Chromatographic Determination of Vitamin C in Aquatic Organisms

A reversed-phase ion-pair liquid chromatographic method using electrochemical detection and internal standardization with isoascorbic acid (IAA) is described for the determination of ascorbic acid (AA) and dehydroascorbic acid (DHAA) in aquatic organisms. Several extractants are compared with regard to recovery and stability of AA and DHAA, including 5% metaphosphoric acid (MPA)-0.54mM ethylenediaminetetraacetic acid (EDTA), 1 % acetic acid (Hac)-1 mM EDTA, 1 % Hac-0.1 % MPA-1 mM EDTA, 2mM homocysteine-1 mM EDTA, and water-methanol (70:30, v/v) containing 0.7mM EDTA. The two nonacidic extractants afford insufficient protection of AA (water-methanol-EDTA) or DHAA (homocysteine-EDTA). High ionic strength acidic mixtures (e.g., 5% MPA) may be associated with a negative drift in the retention times of AA and IAA. Hac (1 %)-EDTA (1 mM) yields lower recovery except when supplemented with MPA. A mixture of 1 % Hac-0.1 % MPA-1 mM EDTA is useful in extracting AA from the brine shrimp Artemia, Brachionus, fish eggs, fish larvae, and postlarvae of shrimp as part of its routine high-performance liquid chromatographic determination in these matrices.

Solid-Phase Extraction and Reversed-Phase Ion-Pair High-Performance Liquid Chromatographic Determination of Chlorophacinone and Diphacinone Residues in Range Grass

A reversed-phase ion-pair liquid chromatographic method is developed for the analysis of chlorophacinone and diphacinone in range grass. Chlorophacinone and diphacinone are extracted from range grass with chloroform, and the extracts are subjected to an aminopropyl solid-phase extraction procedure, concentrated, and analyzed by reversed-phase ion-pair high-performance liquid chromatography. Chlorophacinone and diphacinone are detected by ultraviolet absorption at 285 nm. The average recoveries from range grass fortified at 0.10,1.0, and 10.0 µg/g chlorophacinone and diphacinone are 94% ± 3 and 97% ± 6. The limit of detection for chlorophacinone and diphacinone in range grass is estimated to be 0.015 µg/g and 0.013 µg/g, respectively.

Determination of the stability of morphine tablets by ion-pair reversed-phase liquid chromatography

The main objective of this study was to develop and test the applicability of a sensitive and rapid ion-pair reversed-phase liquid chromatographic method for evaluating a new dosage form for the oral administration of morphine, based on ion pairing with heptane-1-sulphonic acid. Pseudomorphine was identified by gas chromatography-mass spectrometry as the major degradation product of liquid samples of morphine hydrochloride (used as control) that had been subjected to accelerated decomposition. No degradation product was found in the analyses of tablets. Validation of the method yielded a linear regression r=0.9988; the relative standard deviation was 1.10% at 0.24 mg ml −1 (more than 200 samples). The limits of detection were 0.006 mg ml −1 for morphine and 0.012 mg ml −1 for pseudomorphine. The results of stability studies performed during 12 months confirmed the marked stability of tablets prepared in the laboratory.

Determination of metal ions as EDTA complexes by reversed-phase ion-pair liquid chromatography

A reversed-phase ion-pair liquid chromatographic method was developed for the determination of metal ions in aqueous solution. Metal ions were first chelated with EDTA, followed by injection into the chromatographic system. A reversed-phase C 8 column was used to separate the chelate ions with an eluent consisting of acetonitrile-water mixture (8 + 92, v/v) containing 0.002 M tetrabutylammonium as the counter ion. UV detection at 242 nm was applied. Factors affecting the ion-pair formation and the chromatographic separation were studied. This method can be applied to the determination of metal ions such as Pb, Ni, Cd, Fe and Cu. The linear dynamic range is larger than two orders of magnitude, the detection limit is at the ng ml -1 level and the R.S.D. is ca. 3% at mid-calibration range. The method has been successfully used in metal analysis of electroplating waste water. The results are comparable to those of atomic absorption spectrometry.

Separation of nucleotides in homogenates of octopus retina by ion-pair reversed-phase liquid chromatography and identification by mass spectrometry

An isocratic ion-pair reversed-phase liquid chromatographic method has been developed for the determination of thirteen nucleotides including cyclic AMP and cyclic GMP. The resolution capability of this method was evaluated successfully using homogenates of octopus retina, the aim being to elucidate the role of nucleotides (particularly ADP and ATP) in the control of oxidative metabolism. To overcome the inherent lack of specificity of ultraviolet detection we used the coupling of liquid chromatography with mass spectrometry, via a thermospray interface, to confirm the identity of the nucleotides of interest in the biological samples.

Simultaneous determination of levodopa, its main metabolites and carbidopa in plasma by liquid chromatography

An ion-pair reversed-phase liquid chromatographic method for the simultaneous determination of levodopa, 3-O-methyldopa, 3,4-dihydroxyphenylacetic acid, homovanillic acid and carbidopa in plasma designed for clinical trials performed to study the effect of peripheral catechol-O-methyltransferase inhibitors on the metabolism of levodopa is described. The high sample throughput of over 50 samples per day of the method makes it ideal for the assay of the large number of samples encountered in clinical trials. After protein precipitation with perchloric acid the analytes are completely separated within 15 min and determined down to a plasma concentration of 20 ng ml-1 using amperometric detection at 800 mV relative to an Ag/AgCl reference electrode. For all analytes the within-day precision defined as a relative standard deviation (n = 8) is lower than 7 and 3% at plasma concentrations of 20 and 40 ng ml-1, respectively. As the method is specific and highly reproducible, the most important factor affecting accuracy is the stability of the analytes during storage and analysis.

Analysis of Bambuterol Hydrochloride Chemical Reference Substance and Tablets by Liquid Chromatography

Abstract A reversed-phase ion-pair liquid chromatographic method was applied to the analysis of bambuterol hydrochloride chemical reference substance (CRS) and tablets. The purity evaluation of the reference substance was performed with amperometric, UV, diode array and mass spectrometric detection. The total amount of impurities found was less then 0.1%. For the determination of bambuterol hydrochloride in tablets the method reproducibility was 0.9% and the recovery was in the range 99.8–100.7%. Detection limits for conceivable degradation products were about 1 ng. The suitability of different column packing materials was investigated.

Identification and quantification of p-chlorobenzenesulfonic acid in groundwater

Infrared spectra of various extracts were used to identify p-chlorobenzenesulfonic acid in groundwater, and several derivatives were prepared for confirmation. A reversed-phase ion-pair liquid chromatographic method was developed to quantify the compound. Groundwater from the Stringfellow Superfund site was used as a test sample, p-Chlorobenzenesulfonic acid was determined to be the predominant organic species in the water, comprising as much as 50% of the organic carbon.

Statistical optimization of a reversed-phase ion-pair liquid chromatographic method for the analysis of tolmetin sodium in dosage forms

A quantitative liquid chromatographic method in which tolmetin sodium is separated from an internal standard on a C 18 column with detection at 317 nm has been developed with the aid of two statistical optimization procedures. The effects of simultaneously varying the pH, methanol-water ratio, and the concentrations of buffer and ion-pair reagent in the mobile phase were studied. A two-level factorial design was used to investigate the interactions among the variables, and the sequential simplex procedure was used to optimize the separation. A novel quality criterion was developed for the simplex optimization. Using synthetic mixtures, the mean recovery value ± S.D. ( n = 6) of tolmetin sodium was 98.7 ± 0.19% for tablets and 98.5 ± 0.12% for capsules. The assay results for commercial tablets and capsules were comparable to those obtained by the USP XXI procedure.

Ion-pair extraction and high-performance liquid chromatographic determination of tianeptine and its metabolites in human plasma, urine and tissues

An isocratic reversed-phase ion-pair liquid chromatographic method for the determination of tianeptine and its two main metabolites in plasma, urine and tissues, using an internal standard, is reported. The influence of two stationary phases on the retention of the drugs was studied. The drugs were extracted as ion pairs, using a heptane—octanol—tetraheptylammonium bromide mixture (98:2:0.5, v/v/w) as extraction solvent. This extraction procedure yielded plasma drug recoveries of > 60% and allowed UV detection at 220 nm without interference from endogenous components of plasma, urine or tissues. Linear standard curves up to 1.00 μg/ml and drug determination down to 0.01 μg/ml were observed. This method has been successfully applied to the analysis of human plasma and urine samples and of encephales from tianeptine-dosed rats.

High-performance liquid chromatographic method for the determination of amineptine and its main metabolite in human plasma

An isocratic reversed-phase ion-pair liquid chromatographic method for the determination of amineptine and its main metabolite in plasma using an internal standard, is reported. The effects of stationary phase alkyl chain length and the concentration of alkyl sulfonate in the mobile phase were investigated. The drugs were extracted as ion pairs and the influence of various parameters on the extraction efficiency are discussed. Using a heptane-octanol-tetraheptylammonium bromide mixture (98 : 2 : 0.5, v/v/w) as extraction solvent, more than 60% of each drug was recovered with a very good selectivity. UV detection at 220 nm allowed drug determination down to 0.010 μg/ml. Linear standard curves up to 1.00 μg/ml were observed.

Simultaneous determination of amitriptyline, nortriptyline and their respective isomeric 10-hydroxy metabolites in plasma by liquid chromatography

An ion-pair reversed-phase liquid chromatographic method for the determination of the tricyclic antidepressant amitriptyline, its demethylated metabolite nortriptyline, and their respective cis- and trans-hydroxylated metabolites in plasma is presented. After extraction from 1 ml of plasma, the reconstituted residue was chromatographed on a trimethylsilyl packed column using a mobile phase of acetonitrile and acetate buffer with sodium heptane-sulfonate and triethylamine. Recovery of the drugs and its metabolites from plasma ranged from 56 to 99%. The method is suitable for determining plasma concentrations as low as 5 ng/ml (C.V. < 9%) for all six compounds. Plasma concentrations of amitriptyline and its metabolites from eleven different patients are presented.