An HPLC Method for the Determination of Verapamil and Norverapamil in Human Plasma

Abstract

A high performance liquid chromatographic method is presented for the determination of verapamil and its metabolite norverapamil in human plasma. Verapamil and norverapamil are extracted from plasma basified with 0.5M dibasic sodium phosphate (pH 9.5) using ethyl acetate containing trimipramine as an internal standard. A reverse-phase cyanopropylsilane column was used with a mobile phase of 65% acetonitrile and 35% 0.02M acetate buffer (pH 7.0). The minimum detectable limit was 2 ng/ml of plasma. The effect of the pH, molarity, and percent acetonitrile of the mobile phase on the capacity factor was studied. Possible interferences from other drugs administered concurrently are presented.     

An HPLC Method for Determination of Atropine in Human Plasma

Abstract

A development of a high performance liquid chromatographic method for the determination of atropine in human plasma is presented. Atropine is extracted from plasma basified with 0. 1N sodium hydroxide using chloroform, subsequently subjected to base hydrolysis, followed by derivatization of the generated tropic acid with 4-bromomethyl-7-methoxycoumarin (Br-Mmc). The derivative produced has a strong blue fluorescence at excitation wavelength of 328 nm and emission cutoff filter of 389 nm. d1-Mandelic acid as internal standard (I. S.) was added after hydrolysis. The chromatographic separation was achieved on a reversed phase ODS column with a mobile phase of 33% acetonitrile in 0. 01M ammonium phosphate buffer (pH 5). The minimum quantitative limit was 125 ng/ml of plasma.     

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 Ampicillin in Plasma by Paired Ion High Performance Liquid Chromatography

Abstract

A new, accurate, precise, and specific method has been developed for the assay of ampicillin in plasma. No extraction of ampicillin from plasma is called for. Plasma is treated with acetonitrile containing propiophenone as the internal standard, vortexed, centrifuged, and the supernate is injected. A C18 reverse phase column is used. The mobile phase consisted of a mixture of methanol and 0.02M phosphate buffer of pH 6.00, and contained alkyldimethylamine C10 as an ion pair ligand. Detection was by UV at 220 nm. A linear relationship between concentration and peak area ratio was obtained. Recovery, day-to-day, and within-day variation were determined.     

High Performance Liquid Chromatographic Method for Quantitation of a New Antidiabetic Agent in Plasma

Abstract

An HPLC procedure for the detection and quantitation of a new antidiabetic agent, N-(trans-4-isopropylcyclohexylcarbonyl)-D-phenylalanine (A4166), in dog plasma was developed. The drug and internal standard were extracted from plasma using a reversed phase C₁₈ extraction column (Sep-pak). Separation was accomplished on a ERC-ODS-1161 reversed-phase column with a mobile phase of acetonitrile/0.1M phosphate buffer, pH 6.6 (30/70). Quantitation was achieved by monitoring the ultraviolet absorbance at 210 nm. A linear relationship between concentration and peak height ratio (A4166/internal standard) was obtained. The method has been successfully used for analysis of plasma samples from beagle dogs following oral administration of A4166.     

A Simultaneous Determination of Trazodone and Its Metabolite 1-m-Chlorophenylpiperazine in Plasma by Liquid Chromatography with Electrochemical Detection

Abstract

A liquid chromatographic method coupled with electrochemical detection was developed to measure plasma trazodone and its metabolite 1-m-chlorophenylpiperazine (m-CPP). Following extraction from 1 ml of alkaline plasma with methyl-t-butyl ether, the extracts were chromatographed on a reversed phase trimethylsilyl bonded column using a 0.05 M phosphate buffer and acetonitrile (90:10) with n-nonylamine and sodium heptane sulfonate added to the mobile phase. The compounds were detected via a thin layer electrochemical transducer with glassy carbon electrodes at a potential of + 1.15V vs Ag/AgCl reference electrode. The recovery of trazodone ranged from 91–97% and the coefficient of variation was less than 5% for between run and within-run analyses. The recovery of m-CPP ranged from 82–86% and the coefficient of variation was less than 8% for between run and within-run analysis. Steady state plasma concentration data are presented from several patients.     

A Simple Isocratic HPLC Method for the Determination of Metoclopramide in Plasma and Urine

Abstract

Metoclopramide concentrations in plasma and urine were determined by high performance liquid chromatography using a cyanopropylsilane column and UV detection. The mobile phase consisted of 0.03M sodium acetate (pH 7.4) and acetonitrile. The plasma samples were extracted with dichloromethane after pH adjustment. Urine proteins were precipitated with acetonitrile. The reproducibility and precision of the methods were demonstrated by the analysis of samples containing 5 – 200 ng/ml plasma and 0.25 – 200 ug/ml urine.

The glucuronide and sulfate conjugates of metoclopramide were also quantitated after differential acid hydrolysis of urine samples. The conditions for acid hydrolysis were studied. The methods have been applied to the analysis of plasma and urine samples obtained from human volunteers.     

Determination of Belotecan in the Plasma,Bile, and Urine of Rats by High-Performance Liquid Chromatography with Fluorescence Detection and Its Application to a Pharmacokinetic Study

Abstract

A simple and reliable high-performance liquid chromatographic (HPLC) method was developed for the determination of belotecan in the plasma, urine, and bile samples of rats. Belotecan was analyzed with HPLC using a C18 column with fluorescence detector. A mixture of acetonitrile–0.1 M potassium phosphate buffer at pH 2.4 (25:75, v/v) and 0.2% trifluoroacetic acid was used as the mobile phase. The lower limits of quantitation (LOQ) were 5 ng mL^?1 for the plasma and 5 µg mL^?1 for the urine and bile samples. The method has been readily applied for the routine pharmacokinetic study of belotecan in small laboratory animals.     

High Pressure Liquid Chromatographic Analysis and Dissolution of Famotidine in Tablet Formulation

Abstract

A reversed phase high pressure liquid chromatographic method was developed for the quantitation of famotidine in tablet formulation using a mobile phase consisting of 0.1M phosphate buffer (84%), acetonitrile (11%) and methanol (5%) at a pH of 6.5. The detection wavelength was set at 285 nm. The method is precise and adaptable for quality control purposes. The use of the analytical method hi studying tablet dissolution is described.     

Simultaneous Determination of Flunixin,Phenylbutazone, Oxyphenbutazone and γ-Hydroxyphenylbutazone in Equine Plasma by High-Performance Liquid Chromatography: With Application to Pharmacokinetics

Abstract

A high performance liquid chromatographic method was developed for the simultaneous determination of flunixin, phenylbutazone, oxyphenbutazone and γ-hydroxyphenylbutazone in equine plasma. Samples of plasma or sera were deproteinated by addition of acetonitrile containing the internal standard naproxen. The concentration step consisted of taking an aliquot of deproteinated plasma, evaporating under nitrogen to dryness and redissolving in mobile phase. The extracts were chromatographed on a Spherisorb 5 μm ODS column using an isocratic mobile phase of methanol (30% v/v), acetonitrile (20% v/v) and pH 3.0 1% acetate buffer (50% v/v) at a flow rate of 1.2 ml/min using naproxen as the internal standard. The detection limit for flunixin, phenylbutazone, oxyphenbutazone and γ-hydroxyphenylbutazone was 50 ng/ml.

The developed chromatographic method was applied to the determination of equine nonsteroidal anti-inflammatory treatment. Plasma samples from clinically treated horses administered flunixin and phenylbutazone simultaneously are reported. Effect of different anticoagulants used in sampling is reported.     

An Isocratic HPLC Method for the Determination of Cephalosporins in Plasma

Abstract

An isocratic reversed-phase liquid chromatographic method for the determination of eight cephalosporins in human plasma using UV detection at 254 nm is described. Plasma proteins were precipitated using acetonitrile prior to injection of a 10 μl aliquot onto an octadecylsilane column. The mobile phases consisted of 6–11% acetonitrile in sodium dihydrogen phosphate (0.01M). The minimum detectable limit for each drug was less than 1 γg/ml of plasma. Possible interference from other drugs which might be administered concurrently is discussed. The reproducibility and precision of the method for cephalosporin assay are shown from the analysis of plasma containing 5–500 γg/ml of plasma. The chromatographic behavior of the eight cephalosporins was examined by varying mobile phase conditions.     

Rapid Quantitation of Verapamil in Plasma by High-Performance Liquid Chromatography

Abstract

A simple and sensitive liquid chromatographic assay procedure using a fluorescence detector for the quantitative determination of verapamil in plasma without extraction was developed. After precipitating the protein with acetonitrile, the resulting supernatant liquid was injected onto the column for analysis. Chromatographic separation was achieved on C₁₈ reversed phase column and the eluting solvent was the isocratic mixture of methanol, acetonitrile and pH 3.0 glycine buffer (1:4:5). With this mobile phase the drug and its internal standard were well separated from the interference of the plasma sample. The average recovery of verapamil from 3 replicate samples of different concentration (100–600 ng/mL) were 95.5 ± 5.68%. The minimum amount of verapamil detectable by this method was 40 ng/mL of sample. The elimination half-life (β-phase) of this drug in rabbits was found to be 3.7 hours.     

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.     

HPLC Isolation and Characterization of Pentacarboxylic Porphyrins Derived from Uroporphyrinogen III

Abstract

A reversed-phase HPLC system with 22% (v/v) acetonitrile in 1 M ammonium acetate buffer pH 5.16 as mobile phase on an ODS-Hypersil column is developed for the analysis, isolation and characterization of the pentacarboxylic porphyrins derived from uroporphyrinogen III. The results proved conclusively that enzymic decarboxylation of uroporphyrinogen III does not always begin at the ring D acetic acid group and proceeds in a clockwise manner as currently believed.     

Determination Of Serum Cephalexin By High Performance Liquid Chromatography

Abstract

A simple and specific method for the assay of cephalexin in human serum by high performance liquid chromatography is described. Cephalexin was extracted from serum with 5 fold volume of methanol and chromatographed on a reversed-phase column using 30 % aqueous acetonitrile solution containing 0.005 M sodium 2-propanesulfonate (pH 3.0) as the mobile phase. Cephalexin was detected by the absorbance at 254 nm. Only 20 μl of serum was required and all of the operations for analysis were completed within 10 min. This method was proved to be effective in the rapid monitoring of serum cephalexin concentration in humans who received its ordinal and long-acting preparations.     

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.     

Quick and Simple Determination of Dihydroergotamine by High-Performance Liquid Chromatography

Abstract

A simple and rapid liquid chromatographic assay method using a fluorescence detector for quantitation of dihydroergotamine in plasma without extraction was developed. After precipitating the protein with acetonitrile, the supernatant liquid was directly injected for analysis. Chromatographic separation was achieved on C₁₈ reversed phase column and the mobile phase was the isocratic mixture of methanol, acetonitrile and glycine buffer (0.5:3.5:6.0). With this eluting solvent the drug and its internal standard were well separated from the interference of the plasma sample. The average recovery of dihydroergotamine from 6 replicate samples of different concentrations (5-30 ng/ml) were 92.2 ± 3.37%. The minimum amount of dihydroergotamine detectable by this method was 2 ng/ml of sample.     

High-Performance Liquid Chromatography Analysis of Hexamethylmelamine and its Demethylated and Hydroxylated Metabolites in Mice Plasma

Abstract

A selective assay of hexamethylmelamine and its metabolites in mice plasma has been developed. The method uses protein precipitation with acetonitrile in glass centrifuge tubes and analysis of the resulting supernatant by reverse-phase high performance liquid chromatography on a Lichrocart RP 18 column with a mobile phase acetonitrile-phosphate buffer containing n-propylamine (pH 7,5). The influence of various parameters on the separation (solvent composition, pH, n-propylamine content, temperature) has been examined.     

Determination of Danazol in Plasma by High Performance Liquid Chromatography

Abstract

An HPLC method has been developed for the determination of danazol in human plasma. the chromatographic conditions consisted of an ODS column 80 × 7.0 mm, 3 μm; a mobile phase of 71:29, methanol:20 mM potassium dihydrogen phosphate. A UV-Visible detector was set at 288 nm to monitor the danazol peak. Danazol was extracted from plasma with acetonitrile which was salted out with potassium carbonate. Prior to salting out, cadmium sulfate was mixed with the acetonitrile-plasma mixture to remove any interfering constituents. the extracted acetonitrile layer was evaporated and the residue was reconstituted in the mobile phase before injection. the method was found to be reproducible with relative standard deviation (%rsd) from 2.3 to 7.2%. A number of clinically important drugs did not interfere with danazol determination.     

Simultaneous Determination of Caffeine and Antipyrine in Plasma and Saliva using High-Performance Liquid Chromatography

Abstract

A rapid and sensitive method of quantitation of caffeine and antipyrine in plasma and saliva is described. Caffeine, antipyrine and phenacetin, the internal standard, are readily extracted from alkalinized plasma and saliva into dichloromethane. After evaporation of the organic solvent, the residue is analyzed by HPLC using a mobile phase of 25% acetonitrile in 0.02 M phosphate buffer at a flow rate of 1.5 ml/min. and a C18 reverse phase column. Baseline separation of all peaks is achieved with retention times for all compounds of less than 10 minutes. There is no interference from endogenous compounds or metabolites of caffeine or antipyrine.