A Comparison and Clinical Applications of Ammonium Acetate and Phosphate Based Mobile Phases for the Separation of Catecholamines on Reversed Phase Columns

Abstract

A novel ion paired high performance liquid chromatographic system on reversed phase columns with ammonium acetate buffer as eluent is described for the separation of catecholamines. The advantages of using ammonium acetate buffer have been systematically studied and compared with the more widely employed phosphate buffer. The applicability of the method was demonstrated by analysis of catecholamines in clinical specimens.     

High-Performance Liquid Chromatographic Analysis of Theophylline in the Presence of Caffeine in Blood Serum and Pharmaceutical Formulations

Abstract

A reversed phase high-performance liquid chromatographic (HPLC) method has been established for the separation and quantitative determination of the alkaloid theophylline in the presence of caffeine -internal standard- in blood serum and in pharmaceutical preparations. The separation was performed on Spherisorb-5 RP-18 5μm reversed phase column using methanol: 0.038 M ammonium acetate: acetonitrile (38: 57:5) at a pH of about 7.20. The eluted alkaloids are detected at 272 nm. The retention time is 3.09 min for theophylline and 3.85 min for caffeine. The correlation of the integrated peak area with the concentration of theophylline showed a linear relationship between 0.05 to 5.0 theophylline in blood serum, tablets, sprinkels, syrups, suppositories and injectable solutions.     

Rapid Analysis for Codeine by Reversed-Phase High-Performance Liquid Chromatography

Abstract

A reversed phase high-performance liquid chromatographic (HPLC) method has been established for the separation and quantitative determination of the alkaloid codeine in pharmaceutical preparations and in body fluids. The minimum detectable concentrations for body fluids were 5ppb and 7ppb respectively for urine and whole blood with an analysis time of under 5 min. A RP-8 Spheri-5 guard column and a RP-8 Lichrosorb-10 column were used and codeine was detected at its absorption maximum wavelength of 212 nm using an eluting system of methanol: 0.5% w/v aqueous ammonium acetate (70:30) at a pH of about 7.0.     

Optimization of chromatography to overcome matrix effect for reliable estimation of four small molecular drugs from biological fluids using LC–MS/MS

The article describes a systematic study to overcome the matrix effect during chromatographic analysis of gemfibrozil, rivastigmine, telmisartan and tacrolimus from biological fluids using LC–ESI–MS/MS. All four methods were thoroughly developed by the appropriate choice of analytical column, elution mode and pH of mobile phase for improved chromatography and overall method performance. Matrix effect was assessed by post-column analyte infusion, slope of calibration line approach and post-extraction spiking. The best chromatographic conditions established were: Acquity BEH C₁₈ (50 × 2.1 mm, 1.7 μm) column with 5.0 mm ammonium acetate, pH 6.0–methanol as the mobile phase under gradient program for gemfibrozil; Luna CN (50 × 2.0 mm, 3 μm) column with a mobile phase consisting of acetonitrile–10 mm ammonium acetate, pH 7.0 (90:10, v/v) for rivastigmine; Inertsustain C₁₈ (100 × 2.0 mm, 5 μm) column using methanol–2.0 mm ammonium formate, pH 5.5 (80: 20, v/v) as the mobile phase for isocratic elution of telmisartan; and Acquity BEH C₁₈ (50 × 2.1 mm, 1.7 μm) with methanol–10 mm ammonium acetate, pH 6.0 (95:5, v/v) as mobile phase for tacrolimus. The methods were thoroughly validated as per European Medicines Agency and US Food and Drug Administration guidance and were successfully applied for pharmacokinetic studies in healthy subjects.     

Semipreparative High-Performance Liquid Chromatographic Separation of Phosphatidylcholine Molecular Species From Soybean Leaves

Abstract

An improved high-performance liquid chromatographic (HPLC) method using UV detection at 205 nm is described for the semipreparative separation of the molecular species of phosphatidylcholine (PC) from soybean leaves. the separations of PC molecular species are achieved isocratically within ca. 75 min on C 18 reversed-phase column using the mobile phase, methanol:0.1 M ammonium acetate, pH 7.4 (95:5, v/v). Five molecular species for soybean PC are identified as 18:3/18:3, 18:2/18:3, 18:2/18:2, 16:0/18:3 and 16:0/18:2.     

Simultaneous High Performance Liquid Chromatographic Determination of Amitriptyline Hydrochloride and Perphenazine in Tablet Formulations

Abstract

A rapid, precise, and accurate high performance liquid chromatographic procedure is presented for the simultaneous determination of amitriptyline hydrochloride and perphenazine in two component tablet formulations. The related compounds of amitriptyline hydrochloride were separated, making the determination specific for amitriptyline hydrochloride and perphenazine. The method was used for the assay and content uniformity for three commercial products. The mobile phase was 0.02 M ammonium acetate in aceto-nitrile: methanol: water (45:15:40) solution and the pH was adjusted to 5.0 by acetic acid. The column was a supelcosil (5 μm) LC-8-DB (250 mm × 4.6 mm i. d). The method was tested for linearity, recovery, and specificity.     

Determination of Ergotamine Tartarate and Cyclizine Hydrochloride in Pharmaceutical Tablets by Reverse Phase HPLC

Abstract

A high performance liquid chromatographic procedure is presented for the determination of ergotamine tartarate and cyclizine hydrochloride in pharmaceutical tablets. An aliquot of the sample is dissolved in methanol containing ethyl p-hydroxybenzoate as an internal standard and chromatographed on a 5 üm, C₁₈, Hibar pre-packed, Lichrospher (250 mm × 4.0 mm i.d.), column using a mobile phase of 0.01 M ammonium acetate in acetonitrile: water: triethylamine (35:64:1) solution, the pH was adjusted to 3.7 with glacial acetic acid. The method was tested for linearity, recovery, and specificity and was found to be fast, sensitive, accurate and reproducible with a total elution time of six min.     

Determination of Cefmenoxime in Human Serum by Ion-Pair Reverse-Phase High Performance Liquid Chromatography

Abstract

A highly reproducible ion-pair reverse-phase high performance liquid chromatographic assay for cefmenoxime in human serum has been developed. A simple sample cleanup procedure is employed. Cefoxitin is the internal standard and separation is achieved using a C-18 μ-Bondapak column. The mobile phase consists of 20% acetonitrile and 80% 0.05 M ammonium acetate buffer containing 0.005 M tetrabutylammonium hydrogen sulphate as the ion-pair agent. Samples are quantitated by UV detector at 254 nm and 0.02 aufs with an assay sensitivity of 0.625 μg/ml. The method has been successfully applied in a clinical setting.     

High Performance Liquid Chromatography of Enkephalin and Endorphin Peptide Analogs

Abstract

Reverse phase systems are presented which utilize a μ alkylphenyl column and ammonium acetate buffered aqueous acetonitrile mobile phases to separate mixtures of enkephalin and endorphin peptide analogs. High pressure liquid chromatographic separations of mixtures of enkephalin diastereomers as well as mixtures of other closely similar analogs have been developed.

Endorphin analogs were observed to be quite hydrophobic and required mobile phases containing 40% or more acetonitrile for reasonable elution times. The enkephalin analogs by comparison required 20% or more acetonitrile. Detection at both 254 and 280 nm was useful in recognizing the important peaks in the elution profile.     

Quantitative High Performance Liquid Chromatographic Determination of Bamipine Combined with Tricyclic Antidepressants and/or Antipsychotics in Pharmaceutical Formulations

Abstract

An advanced reserved phase High Performance Liquid Chromatographic (HPLC) method using UV detection, at 251, is presented for the simultaneous determination of bamipine combined with tricyclic antidepressants and/or antipsychotics in various pharmaceutical formulations. Sample analyses were performed on a bonded reserved-phase C-2,5 μm, 250 × 4.6 mm ID (Lichrosorb RP-2)column using aqueous ammonium acetate at constant ionic strength 0.015 M and acetonitrile 35: 65 as eluent, at a flow rate of 0.9 ml/min. The pH was adjusted to 4.7 with acetic acid. The presence of acetate buffer both shortens the elution time and improves the symmetry of the chromatographic peaks. The retention time was for bamipine, prochlorperazine, trifluoperazine, thioridazine, chlorprothixene, haloperidol, clomipramine, trimipramine 8.83, 11.83, 11.50, 11.08, 10.42, 8.05, 10.25, 9.67 min respectively. Linearity and precision data have been assessed. The method also involves an investigation of the dependence of the capacity ratio k′ on the pH and the polarity of the mobile phase.     

Ionic liquids as mobile phase additives in high-performance liquid chromatography with electrochemical detection: Application to the determination of heterocyclic aromatic amines in meat-based infant foods

The beneficial effects of several ionic liquids (ILs) as mobile phase additives in high-performance liquid chromatography with electrochemical detection for the determination of six heterocyclic aromatic amines (HAs) have been evaluated for first-time. The studied ionic liquids were 1-butyl-3-methylimidazolium tetrafluoroborate (BMIm-BF₄), 1-hexyl-3-methylimidazolium tetrafluoroborate (HMIm-BF₄) and 1-methyl-3-octylimidazolium tetrafluoroborate (MOIm-BF₄). Several chromatographic parameters have been evaluated in the presence or absence of ILs, or using ammonium acetate as the most common mobile phase additive, with three different C18 stationary phases. The effect of the acetonitrile content was also addressed. In general, best resolution, lower peak-widths (up to 72.1% lower) and lower retention factors are obtained when using ILs rather than ammonium acetate as mobile phase additives. The main improvement was obtained in the baseline noise, being 360% less noisy for BMIm-BF₄, 310% for HMIm-BF₄, and 227% for MOIm-BF₄, when compared to ammonium acetate at +1000 mV. Different chromatographic methods using the best conditions for each IL were also evaluated and compared. Finally, the best chromatographic conditions using 1 mM of BMIm-BF₄ as mobile phase additive, the Nova-Pak^® C18 column, 19% (v/v) of acetonitrile content in the mobile phase, and +1000 mV in the ECD, have been applied for the chromatographic analysis of six HAs contained in meat-based infant foods. The whole extraction method of meat-based infant foods using focused microwave-assisted extraction and solid-phase extraction has also been optimized. Extraction efficiencies up to 89% and detection limits ranged between 9.30 and 0.165 ng g⁻¹ have been obtained under optimized conditions.     

Analysis of Dexamethasone Acetate in Pharmaceutical Formulations by HPLC

Abstract

A rapid and effective high-pressure liquid chromatographic method has been developed for the quantitative determination of dexamethasone 21 acetate in pharmaceutical formulations. Sample preparation employs a simple extraction procedure and analysis is carried out on a reversephase chromatographic system using a LiChrosorb RP 18 column and a water-acetonitrile as mobile phase. The extraction procedure gives quantitative recovery and chromatographic results show that drug levels of as 0.1 ppm can be conveniently analyzed without significant background interferences.     

High-Performance Gel Permeation Chromatography of Pectins

Abstract

A high-performance gel permeation chromatographic (GPC) method was developed to determine the molecular weight distribution of pectins. The chromatographic system consisted of a hydrophilic coated silica (SynChropak) as the packing and a pH 3.7 acetate buffer as the mobile phase. By use of this system, high-methoxy, low-methoxy, and amidated pectins could be analyzed within fifteen minutes. By determining partition coefficients (K_d) of pectins as a function of mobile phase composition, K_d values were found to be independent of ionic strength from 0.055 to 0.7 M using pH 3.7 acetate buffers, which was in agreement, with intrinsic viscosity data.     

High Performance Liquid Chromatographic Determination of Flurbiprofen in Pharmaceutical Dosage Forms

Abstract

A rapid, specific and reliable high performance liquid chromatographic assay of flurbiprofen in dosage forms has been developed. Reversed-phase chromatography was conducted using a mobile phase of 0.05 M ammonium acetate and acetonitrile, (40% v/v) PH 5.2 and detection at λ 247 nm. The recovery and coefficient of variation from six placebo tablets spiked with 100 mg of flurbiprofen were 100.1% and 0.4% respectively. Replicate regression analyses of three standard plots in the concentration range 0.5 - 9 mcg/ml obtained on three different days gave a correlation coefficient (0.99996) and the coefficient of variation of the slopes 0.159%. The assay was precise within day and between days as indicated by ANOVA test. It is suggested that the proposed HPLC method should be used for routine quality control and dosage form assay of flurbiprofen.     

Determination of Benzodiazepines in Serum by High Performance Liquid Chromatography Using Radially Compressed Columns and an Aqueous Methanolic Mobile Phase

Abstract

Diazepam, oxazepam and N-desmethyldiazepam are determined by high performance liquid chromatography using a radially compressed C18 column and an aqueous methanolic mobile phase. The chromatographic separation is completed within 10 minutes. The drugs are recovered from serum by extraction with hexane:ethyl acetate 70:30, v/v.

The method is linear in the range 50-1600 ng/ml for all the drugs, Coefficients of variation are less than 6.2% for two studied concentration levels.     

Liquid Chromatographic Analysis for Flecainide with Use of a Microbore Column and Ultraviolet Detection

Abstract

We describe a simple, isocratic high-performance liquid chromatographic method for measuring the oral antiarrhythmic agent flecainide acetate in serum or plasma. Sample analysis involves a simple organic extraction followed by chromatography on a microbore reverse phase column with ultraviolet detection at 298 nm.     

Chromatography of AC-ASP-TYR-MET-GLY-TRP-MET-ASP-NH2 on the Horizontal flow-Through Coil Planet Centrifuge and the High-Speed Multi-Layer Coil Planet Centrifuge

Abstract

The peptide Ac-Asp-Tyr-Met-Gly-Trp-Met-Asp-NH₂ was purified by countercurrent chromatography in the horizontal flow-through coil planet centrifuge. The solvent system used was ammonium acetate, pH 8.5 and n-butanol (1:1 by volume). The high pH served to maintain the peptide in solution. When the upper phase was utilized as the mobile phase better separation of the peptide from impurities resulted. The peptide was also chromatographed in a new apparatus, the high-speed multi-layer coil planet centrifuge. With the lower phase mobile and at a higher temperature, the peptide was fractionated very rapidly in 30 min compared to 7 hr on the other instrument.     

Thin-layer chromatographic fingerprinting of the nonvolatile fraction extracted from the medicinal herb Cistus incanus L.

ABSTRACT

The aim of this study was to provide the first from-start-to-end thin-layer chromatographic method of fingerprinting the Cistus incanus L. raw herbal material, with a purpose to further use it for rapid screening, authentication, and quality control of the traded C. incanus L. herbs. To this effect, 12 different C. incanus L. samples purchased as herbal teas from a local market were extracted by means of the accelerated solvent extraction (ASE) with chemometrically optimized solvent extraction mixture and temperature (methanol–water, 27:73, v/v; 130°C), to derive the polar fraction from the plant samples. Then, the extracts were developed in two thin-layer chromatographic systems, both using the commercially precoated silica gel 60 chromatographic plates, yet two different mobile phases (mobile phase 1, ethyl acetate–formic acid–acetic acid–water, 100:11:11:13, v/v/v/v, and mobile phase 2, ethyl acetate–dichloromethane–formic acid–acetic acid–water, 100:10:10:10:11, v/v/v/v/v). The chromatograms were densitometrically scanned in the reflectance mode at the wavelength λ?=?366?nm to obtain fingerprints of the extracts derived from individual C. incanus L. samples. Mobile phase 2 performed slightly better, because with its use, the maximum number of 11 peaks could be seen in the respective fingerprints, whereas with mobile phase 1, the maximum number of 10 peaks only. Then an antioxidant potential of the investigated herbal extracts was assessed, making use of mobile phase 2 and the 0.20% methanol solution of 2,2-diphenyl picrylhydrazyl as a visualizing reagent. The resulting chromatograms were densitometrically scanned in the extinction mode at the wavelength λ?=?550?nm to obtain biological fingerprints of the extracts. Finally, chromatographic and biological fingerprints underwent a semiquantitative evaluation in terms of the contents of the extracted polar fraction and an overall antioxidant potential of the individual plant species.     

Adsorption of Tetralkyl Ammonium Ions on Reversed-Phase HPLC Columns

Abstract

A systematic study of the influence of several chromatographic parameters upon adsorption of tetralkyl ammonium ions on reversed-phases has been made. Results show that organic solvent content in mobile phase, tetralkyl ammonium hydrophobicity and tetralkyl ammonium concentration in eluent are the most influent variables. A mathematical expression to calculate the adsorbed ion concentration for different combinations of these variables is deduced. Results from this study can be applied in Ion Pair Reversed-Phase Chromatography to calculate column equilibration times, for setting gradient elution separation methods and for the study of retention mechanisms.     

Determination of Ibandronate and its Degradation Products by Ion-Pair RP LC with Evaporative Light-Scattering Detection

A simple method has been developed for analysis of ibandronate and related substances by ion-pair reversed-phase high-performance liquid chromatography (RPIC) with evaporative light-scattering detection (ELSD). After optimization of the chromatographic conditions satisfactory separation of the compounds was achieved on an Intersil C₈ column with an isocratic mobile phase—8:4:88 (v/v) acetonitrile–methanol–12 mM ammonium acetate buffer containing 35 mM n-amylamine (pH 7.0). The mobile phase flow rate was 1.0 mL min^?1. The calibration plot was linear in the range 352 to 1760 µg mL^?1 for ibandronate. The precision and reproducibility were 0.3% and 0.5%, respectively. The average recovery of ibandronate was 100.4% and RSD was 0.6%. The method was validated and shown to be precise, accurate, and specific for assay of ibandronate in bulk material and dosage forms. The proposed liquid chromatographic method can be satisfactorily used for quality control of ibandronate.