Determination of the Antineoplastic Agent Tiazofurin in Plasma by High Performance Liquid Chromatography

Abstract

A simple and sensitive sample clean up procedure and a reverse phase high performance liquid chromatographic assay for tiazofurin in dog plasma has been developed.

Thymidine was used as the internal standard and the elution solvent was 0.16 M acetic acid. The retention times of tiazofurin and thymidine at an elution rate of 2.0 ml/minute were 8.7 minutes and 16 minutes respectively. The assay sensitivity was 1.0 μG/ml. The within run coefficient of variation of 2.0 μG/ml and 20 μG/ml plasma samples were ±7.49% and ±3.37% respectively and the between run coefficient of variation of 2.0 μG/ml and 20.0 μG/ml plasma samples were ±10.46% and ±5.92% respectively.     

A Simple Isocratic High-Performance Liquid Chromatographic (HPLC) Determination of Naproxen in Human Plasma using a Microbore Column Technique

Abstract

A simple and sensitive HPLC method was developed for the determination of naproxen in human plasma. The assay employs a microbore column packed with a C18 reversed-phase material (5 μm ODS Hypersil) with an isocratic mixture of acetonitrile and 10 mM phosphate buffer, pH 2.5 (40:60, v/v) as the mobile phase. The mobile phase was pumped at a flow rate of 0.5 ml/min. For sample analysis 200 μl of acetonitrile containing internal standard (flurbiprofen) was added to 100 μl of plasma. After centrifugation 10 mM phosphate buffer, pH 7.4 (200 μl) was added to the tube, then vortexed and centrifuged. The supernatant (20 μl) was injected onto the HPLC column. The chromatographic separation was monitored by a fluorescence detector at an emission wavelength of 350 nm with an excitation wavelength of 225 nm. The direct precipitation of plasma protein using acetonitrile gave a good recovery for both naproxen and the internal standard. The detection limit was 0.1 μg/ml for naproxen. The intra- and inter-assay coefficients of variation at different concentrations evaluated were less than 10%.     

Determination of Arteether in Plasma Using a Simple and Rapid High-Performance Liquid Chromatographic Assay

Abstract

A simple and rapid high-performance liquid chromatographic (HPLC) assay for the determination of the antimalarial drug arteether in plasma was developed and validated in this report. Perchloric acid was used in this method as a plasma protein precipitant and to attain an acidic medium suitable for the decomposition of arteether to a derivative possessing UV absorption. This derivative and the internal standard (progesterone) were separated from the plasma on a 10 μm μ-Bondapack C₁₈ reversed-phase column at ambient temperature with a mobile phase composed of acetonitrile:water (60:40 v/v) and at a flow rate of 1.5 ml/min. The effluent was monitored at 254 nm with a UV detector. Linear relation between drug concentrations and peak height ratios of arteether derivative to the internal standard was achieved in the range of 0.25-10 μg/ml arteether with a detection limit of 50 ng/ml arteether in plasma. The within-day and between-days precisions were evaluated using 3 different concentrations of arteether. The values of the coefficients of variation were 1.35-1.68% and 1.65-2.82% for within-day and between-day, respectively. This method was applied to determine some pharmacokinetic parameters of arteether after intramuscular injection of 50 mg/kg arteether oily solution to rabbits.     

Stability Indicating HPLC Analysis of Dibromodulcitol in Aqueous Solutions

Abstract

A stability-indicating HPLC analytical method for the anticancer agent dibromodulcitol (DBD, Mitolactol, NSC-104800) has been developed that will completely resolve the compound from its degradation products. A 5 μm octadecylsilane analytical column was used in conjunction with a refractive index detector, with a mobile phase of 98:2 water/methanol. The limit of DBD detection was determined to be 250 ng (5 μg/ml) with a signal to noise ratio of 2:1. Intraday variation of the method, as percent relative standard deviation, was 4.37% (12.5 μg/ml) and 0.973% (250 μg/ml), and interday variation was 3.93% (250 μg/ml). Comparison with potentiometric titration of bromide after digestion of DBD with NaOH indicated that the method was more sensitive and specific than titration. The method has been used in tablet content analysis, as well as degradation studies of DBD in solution.     

HPLC Determination of Xylazine in Equine Plasma by High Performance Liquid Chromatography

Abstract

A high performance liquid chromatographic (HPLC) method is described for the determination of xylazine in equine plasma. The drug and internal standard (pindolol) were separated on a 5 μm cyanopropyl-modified column (250 × 4.6 mm i.d.) using a buffer-acetonitrile mixture containing an ion pairing reagent. The drug and internal standard were isolated from plasma by liquid extraction into ethyl acetate. The method was validated over the concentration range 50–2000 ng/ml in plasma; the reproducibility, expressed as the mean co-efficient of variation was less than 5.0% for both between-day and within-day replicate determinations. The method was linear over the concentration range studied. No interferences were observed from endogenous plasma components and the limit of detection was 20 ng/ml. The method was successfully applied to the determination of xylazine in equine plasma in a crossover study design for pharmacokinetic measurements.     

High Performance Liquid Chromatographic Determination of Oxamniquine in Plasma Samples

Abstract

A sensitive and reliable liquid chromatographic assay procedure for the quantitation of oxamniquine in plasma or urine was developed. Chromatographic separation was achieved on a reversed-phase phenyl colum using U.V. Detection at 254 nm. The eluting solvent was the mixture of 0.05 M acetate buffer pH 5 and acetonitrile (3:7). With this mobile phase the drug and its external standard were well separated from the interference of the blank samples. The average recovery of oxamniquine from 3 or more replicate dog plasma samples of different concentration (0.125 ? 4.00 μg/ml) was 95.5% and its coefficient of variation was 4.17%. The reproducibility of the assay was confirmed by the analysis of variance test for the slopes of the three standard plots obtained from plasma samples at three different occasions (F=4.2, p > 0.01). The detection limit for plasma samples was approximately 20 ng/ml. The method was applied to measure the plasma level vs, time profile of this drug following a single bolus intravenous dose of 16 mg/kg to a dog.     

Measurement of Cefixime in Serum and Cerebrospinal Fluid by High-Performance Liquid Chromatography

Abstract

Cefixime is a new cephalosporin antibiotic for oral administration. A high-performance liquid chromatographic (HPLC) method was developed to measure cefixime in small volumes of serum and cerebrospinal fluid (CSF) to conduct a pharmacokinetics study in pediatric patients. The assay involved precipitation of serum proteins with 6% trichloroacetic acid, using 7-hydroxycoumarin as an internal standard. Chromatographic separation was accomplished using ultrasphere C8 column and mobile phase containing 15% acetonitrile in a buffer at a detection wave length of 280 nm. The retention time of cefixime and 7-hydroxycoumarin was about 5 and 9 minutes, respectively. The method was suitable for quantitation of cefixime at a concentration ranging from 0.05 to 10 μg/ml. The coefficient of variation was less than 3%. The technique was used successfully to measure cefixime in serum and CSF obtained from an infant receiving cefixime.     

Determination of Fleroxacin in Plasma by Direct Injection High Performance Liquid Chromatography with Column Switching

ABSTRACT

A high-performance liquid chromatography (HPLC) assay was developed for the determination of fleroxacin in plasma. The plasma samples were directly introduced onto a HPLC column after filtering through a MolcutII® membrane filter, which removes high molecular weight proteins. The fleroxacin in filtrate was separated from interfering substances and retained on a pre-column using an ODS stationary phase and then was introduced to an analytical column with an ODS stationary phase by column switching. Fleroxacin and lomefloxacin, as an internal standard, were detected by ultraviolet absorbance at 295 nm. Determination of fleroxacin was possible over the concentration range 50-4000 ng/ml; the limit of detection was 20 ng/ml. The recovery of fleroxacin added to plasma was 97.3-100.4% with a coefficient of variation of less than 2.2%. This method is applicable to drug level monitoring in the plasma of patients being treated with fleroxacin and of healthy volunteers participating in pharmacokinetic studies.     

An Improved Method for the Determination of Reserpine in Plasma Using Liquid Chromatography with Fluorescence Detection

Abstract

A liquid chromatographic method coupled with fluorescence detection was developed to measure plasma reserpine concentrations. After extraction from 3 ml of plasma, the reserpine and internal standard (methyl-18-triethoxy benzoyl reserpate) residues were oxidized to their respective fluorophors by vanadium pentoxide and chromatographed on a reversed phase trimethylsilyl column. These compounds were detected at excitation wave length 460 nm and analyzed at 570 nm. The minimum quantifiable level was ca 0.3 ng/ml and the absolute recovery was determined to be between 78–83%. The coefficient of variation was less than 9% for day-to-day and within run analyses. This method is suitable for pharmacokinetic studies of reserpine in man.     

High Performance Liquid Chromatographic Determination of Amiodarone in Plasma and Tissues

Abstract

A simple and rapid high-performance liquid chromatographic (HPLC) method for the determination of amiodarone (AD) in plasma and tissues was developed. The method involved deproteinization of plasma or homogenized tissue with acetonitrile containing an internal standard (N-Cetylpyridinium chloride) followed by reversed phase chromatography using μ bondapack C₁₈ column (10μm) with a mobile phase consisting of acetonitrile - methanol - sodium dihydrogen phosphate buffer (70:10:20%, v/v), the pH adjusted to 4.0 and pumped at flow rate of 1.0 ml/min. The column effluent was monitored at 242 nm. A linear relationship was obtained between peak height ratios (drug to internal standard) versus drug levels over the concentration range of 50–750 ng/ml. The detection limit of AD in plasma and tissues by this method was 20 ng/ml.     

A HPLC Method for the Determination of Butaperazine in Solutions,Tablets, Plasma and Bile

Abstract

A specific HPLC method for the determination of the antipsychotic drug butaperazine (B) in solutions, tablets, plasma and bile has been developed. The instrument used was a Waters HPLC equipped with a Model 440 Spectrometer and a μ-Bondapak-NH₂ column. The mobile phase, chloroform-methanol (100:3.5) was pumped at a rate of 1.5 ml per minute. Ultraviolet absorbance at a wave length of 280 nm was used for detection. The procedure involved the extraction of the drug from the dosage forms with chloroform and from plasma and bile with hexaneisopropanol (9:1). Hydrocortisone acetate was used as the internal standard. Retention times of 2.4 and 3.9 minutes were obtained for the internal standard and B respectively. Analytical calibration yielded a linear relationship from 0.1–25 μg per ml, with r² value of 0.99. The percentage recovery of B averaged 99% from the dosage forms and 94% from the biological fluids. An improvement in the method for determining B in bile and plasma was later developed. It involved the use of a different mobile phase and detecting the drug fluorometrically.     

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.     

High Performance Liquid Chromatography Determination of Pcmx In Blood Plasma Using Electrochemical Detection

Abstract

A rapid and specific high performance liquid chromatographic (HPLC) procedure has been developed for the determination of p-chloro-mxylenol (PCMX) in blood plasma. the method is based on the extraction of PCMX from plasma with benzene in the presence of a known amount of dichloro-m-xylenol (internal standard). the benzene extract is evaporated to dryness and the residue dissolved in 200 μl of mobil phase. the HPLC system consisted of a reversed-phase column and a 65% methanol:35% ammonium carbonate 0.05% solution as a mobile phase. an electrochemical (EC) detector/glassy carbon electrode set a potential of +0.9V versus Ag/AgCl/3M NaCl is used to monitor the drug. the recovery of PCMX is approximately 98%, the limit of quantitation is 2 ng/ml of plasma for the HPLC-EC procedure. the coefficient of variation is 5.1% over the range of 10-1000 ng/ml of plasma. Data are presented to illustrate the practicality of this method for evaluation of PCMX plasma levels after a single intravenous administration of 500 mg dose of PCMX to five mongrel dogs.     

High Performance Liquid Chromatographic Assay of Phenacemide in Tablets

Abstract

A rapid, specific and reliable high-performance liquid chromatographic assay of phenacemide in tablets has been developed. Reversed-phase chromatography was conducted using a mobile phase of acetonitrile and acetate buffer, pH 4.2 (50% V/V) and detection at 254 nm. The recovery and coefficient of variation from six placebo samples of 100 mg were 100.2% and 0.57%, respectively. The percent recovery of 10 replicate commercial tablets was 101.1% of the label amount and its coefficient of variation was 0.95%. Regression analyses of three standard plots in the concentration range of 15-150 μ g/ml obtained on three different days gave a correlation coefficient > 0.999 and the coefficient of variation for their slopes <2%. The HPLC method is rapid as it takes - 1 hour to analyze six commercial tablets compared with 6 hours consumed by the USP method. The assay was precise within day and between days as indicated by ANOVA test.     

Stability-Indicating High Performance liquid Chromatographic Determination of Diltiazem Hydrochloride

Abstract

A rapid, selective and accurate high-performance liquid chromatography method (HPLC) for the determination of diltiazem hydrochloride using clonazepam as internal standard is developed. The investigated compounds were eluted from 5 u Micropak MCH-5 reversed phase column at ambient temperature with a mobile phase comprising of acetonitrile-water (48:52%) at pH 3.3 and containing 0. 01M sodium-n-octane sulfonate as an ion pairing substance. The mobile phase was pumped at a flow rate of 1 ml min^?1 and the effluent was monitored at 239 nm. The retention times of the internal standard and diltiazem were at 3 min and 9 min, respectively. A linear relationship was derived between the peak height ratio (diltiazem/clonazepam) and diltiazem concentration over the range 1–10 μg ml^?1. Detection limit for the drug is 0.2 μg ml^?1 at 0.01 aufs. The developed procedure was applied to study the stability of diltiazem in the presence of its degradation products as well as in the presence of formulation excipients.     

A Simple High-performance Liquid Chromatographic Assay for Loracarbef in Human Plasma

Abstract

A simple, rapid and reproducible high-performance liquid chromatographic (HPLC) method for the determination of loracarbef in human plasma has been developed and evaluated. Plasma protein was precipitated with ammonium sulfate. The drug and the internal standard (Cefetamet) were eluted from a μ-bondapak C-18 column with a mobile phase consisting of acetonitrile:methanol:water:glacial acetic acid (2.5:17.5:79.2:0.8%, v/v). The column eluent was monitored at 265 nm. Quantification was achieved by the measurement of the peak-height ratio of the analyte to the internal standard and the limit of quantification for loracarbef in plasma is 0.5 ug/ml. The within-day coefficient of variation (CV) ranged from 2.28% to 3.67%, and between-day CV from 2.38% to 5.59% at three different concentrations. The absolute recoveries ranged from 91.1% to 93.88%, and the relative recoveries from 93.4% to 108% at three different concentrations. Preliminary stability tests showed that loracarbef is stable for at least 5-weeks in human plasma after freezing. The method is applied for the determination of the pharmacokinetic parameters of loracarbef after oral administration to 2 beagle dogs.     

Sensitive high-performance liquid chromatographic determination of famotidine in plasma. Application to pharmacokinetic study.

A sensitive high-performance liquid chromatographic (HPLC) method for the quantitation of famotidine in human plasma is described. Clopamide was used as the internal standard. Plasma samples were extracted with diethyl ether to eliminate endogenous interferences. Plasma samples were then extracted at alkaline pH with ethyl acetate. Famotidine and the internal standard were readily extracted into the organic solvent. After evaporation of ethyl acetate, the residue was analysed by HPLC. The chromatographic separation was accomplished with an isocratic mobile phase consisting of acetonitrile-water (12:88, v/v) containing 20 mM disodium hydrogenphosphate and 50 mM sodium dodecyl sulphate, adjusted to pH 3. The HPLC microbore column was packed with 5 microns ODS Hypersil. Using ultraviolet detection at 267 nm, the detection limit for plasma famotidine was 5 ng/ml. The calibration curve was linear over the concentration range 5-500 ng/ml. The inter- and intra-assay coefficients of variation were found to be less than 10%. Applicability of the method was demonstrated by a bioavailability/pharmacokinetic study in normal volunteers who received 80 mg famotidine orally.     

High Performance Liquid Chromatographic Assay of Methoclopramide in Plasma Using a Silica Gel Column and An Aqueous Meobile Phase

Abstract

A high performance liquid chromatographic method for quantitating matoclopramide in plasma is presented. Proteins ara precipitated from the plasma sample with acetonitri la containing the internal standard, procainamida. The treated samples ara than analyzad using an Ultrasphere Si column, an aqueous solution at pH 7 of 65% CH³CN and 5.0 mM (NH₄)₂HPO₄ as a mobile phase, and a fluorescence detector. The retention times for drug and intarna1 standard ara 11.2 and 13.2 min, respectively. The caibration curve is Linear from 0.89 to 44.5 ng/ml. The detection limit is 0.89 ng/ml [signaL/hoisa = 31] for 0.2 ml plasma samples Pracision is measured by intraday and intarday coefficients o f variation, which are less than 10%. This method is currently being used for pharmacokinetic studies of methoclopramide.     

Analysis of Plasma Diethyldithiocarbamate–A Metabolite of Disulfiram

Abstract

A reversed-phase high-performance liquid chromatographic analysis was developed for diethyldithiocarbamate in plasma. Following treatment of plasma (1 ml) with methyl iodide (250 μl), biphenyl (internal standard, 1.8 μg) was added in chloroform (6 ml). After shaking (30 min.), the chloroform was separated and evaporated under nitrogen (to 50 μl). Acetonitrile (250 μl) was added and the solution was again evaporated under nitrogen (to 100 μl). Aliquots (25 μl) were chromatographed using acetonitrile: acetate buffer (65:35, pH 4) at 2.5 ml/min on a 5 micron C-8 column with detection at 276 nm. Recovery of methyldiethyldithiocarbamate (MeDDC) was 92.5 ± 3.2%. Retention times and theoretical plates for MeDDC and biphenyl were 3.0 and 4.6 min., and 4660 and 6336 respectively. The analysis was linear over the range 25 to 400 ng/ml with a coefficient of variation of 3.2%. Analysis of samples after intravenous disulfiram (10 mg) administration to rats yielded a total body clearance of 343 ml/min. This supports the view that metabolism is principally by extra-hepatic routes.     

RP‐HPLC Method for the Determination of Tenofovir in Pharmaceutical Formulations and Spiked Human Plasma

Abstract

A simple reverse‐phase high‐performance liquid chromatographic method for the determination of tenofovir disoproxil fumarate (TDF) in pharmaceutical formulations and human plasma samples has been developed and validated. Piroxicam (PRX) was used as an internal standard. The assay of the drug was performed on a CLC C₁₈ (5 μ, 25 cm×4.6 mm i.d.) with UV detection at 259 nm. The mobile phase consisted of acetonitrile–water mixture in the ratio of 75∶25, and a flow rate of 1 ml/min was maintained. The standard curve was linear over the range of 0.2–10 µg/ml (r ²=0.9966). Analytic parameters have been evaluated. Within‐day and between‐day precision as expressed by relative standard deviation was found to be less than 2%. The method has been applied successfully for the determination of TDF in spiked human plasma samples and pharmaceutical formulations.