High-performance liquid chromatography separation of chlordecone and its metabolites

Summary High-performance liquid chromatography (HPLC) was used to separate chlordecone (Kepone), and two of its metabolites, hydrochlordecone and dihydrochlordecone. Elution of the three peaks occured after the solvent concentration reached 100% methanol and was maintained at 100% for approximately five minutes. The method was linear for chlordecone in the concentration range of 10 to 100 g.     

High-performance liquid chromatography of microbial acid metabolites

The use of high-performance liquid chromatography with a cation-exchange column and effluent monitoring at 210 nm has been evaluated for the profiling of selected microbial metabolites including aliphatic, dicarboxylic, and phenolic acids, as an adjunct to the identification of selected bacteria, detection of bacterial metabolites in foods, and the monitoring of industrial microbial fermentations. Advantages of the technique include the simultaneous profiling of different classes of organic acids without derivatization. Most applications require only qualitative or semi-quantitative data. For others, data are given on the day-to-day reproducibility for several acids.     

High-performance liquid chromatography of levomepromazine (methotrimeprazine) and its main metabolites

The phenothiazine drug levomepromazine (methotrimeprazine) has five metabolites which previously have been identified in plasma from psychiatric patients. These are formed by sulphoxidation, N-demethylation, O-demethylation and aromatic hydroxylation in two different positions. A high-performance liquid chromatographic system is described for the analysis of levomepromazine and its main metabolites on a Supelcosil C18-DB column, based on ion-pair formation with sodium docecyl sulphate. The effects of variations in pH, buffer concentration, counter-ion concentration, temperature and concentration and composition of the organic solvent were examined. The six components may be analysed in 27.4 min at room temperature using 25 mM sodium dodecyl sulphate in 500 mM ammonium acetate buffer (pH 5.0)-5% v/v tetrahydrofuran in acetonitrile (50:50, v/v) as the mobile phase.     

High-performance liquid chromatographic assay of betamethasone 17-valerate and its degradation products

A high-performance liquid chromatographic assay of betamethasone 17-valerate is described. The procedure may be use for quantitative assay of the degradation products, betamethasone 21-valerate and betamethasone, and the application to the analysis of ointments is described. The method is also suitable for the determination of the kinetics of decomposition from one experimental run, and the determination of rate constants from a four-compartment sequential reaction is described. The procedure is also applicable to other corticosteroids, and hydrocortisone 17-butyrate, hydrocortisone 21-butyrate, and hydrocortisone may similarly be determined without modification to the method.     

Determination of estradiol and its degradation products by liquid chromatography

A novel HPLC method for simultaneous determination of estradiol and its seven degradation products in topical gel was developed. Zorbax SB-CN (150 mm x 4.6 mm, 5 microm) analytical column and mobile phase composed of acetonitrile, phosphoric acid 0.085%, and tetrahydrofurane (27:63:10, v/v/v) at flow-rate 1.0 ml min(-1) were used for the chromatographic separation using UV detection at 225 nm. The active substance estradiol was separated from all its known degradation products successfully. Two degradation products estrone and Delta(9(11))-estrone were not separated sufficiently, their peaks were evaluated as a sum of two components. The method was validated according to ICH guideline recommendations and thereafter it was successfully applied for stability tests of topical cream Estrogel HBF in the quality control laboratory. Limits of detection for degradation products ranged from 1.03 x 10(-5) to 1.14 x 10(-4) mg ml(-1), limits of quantitation for degradation products were in the range 3.43 x 10(-5) to 3.81 x 10(-4) mg ml(-1). The developed method is selective, precise, accurate and sensitive enough for determination of estradiol and its known degradation products.     

High-performance liquid chromatography of pefloxacin and its main active metabolites in biological fluids

We describe a high-performance liquid chromatographic (HPLC) method for the analysis of pefloxacin, a new antibacterial agent, in plasma and urine following administration of a therapeutic dose in humans. HPLC assay of pefloxacin and its two main active metabolites in urine is also described. The applicability of the methods to pharmacokinetic studies of pefloxacin in humans is demonstrated.     

High-performance liquid chromatographic analysis of glutathione and its thiol and disulfide degradation products

A rapid and sensitive high-performance liquid chromatographic method for quantitation of picomole levels of glutathione, glutathione disulfide, cysteine, cystine, cysteinylglycine, cysteinylglycine disulfide and cysteine glutathione-mixed disulfide in biological samples is described. The compounds were separated isocratically on a reversed-phase column by ion-pair chromatography. The mobile phase consisted of an aqueous buffer containing 0.1 M monochloroacetic acid and 3.3 mM 1-heptanesulfonic acid (pH 2.60)-methanol-N,N-dimethylformamide (96.5:3.0:0.5). After chromatographic separation, the disulfides were reduced by a potential (-1.0 V) from a battery, with subsequent detection of all thiols by electrochemical oxidation (+0.15 V) with a dual gold-mercury electrode. Thiol and disulfide concentrations were determined in tissue extracts (liver and kidney) and fluids (bile and plasma) from control rats and rats treated with acivicin, an inhibitor of gamma-glutamyltranspeptidase. A marked increase in biliary glutathione concentration was observed in treated animals with a corresponding decrease in cysteine and cysteinylglycine concentrations. The results demonstrate that this method is useful for measuring glutathione and its degradation products in tissues and fluids.     

High-performance liquid chromatographic determination of the polar metabolites of nifedipine in plasma, blood and urine

A relatively simple reversed-phase high-performance liquid chromatographic method for the determination of the polar metabolites of nifedipine in biological fluids is described. After conversion of 2-hydroxymethyl-6-methyl-4-(2-nitrophenyl)pyridine-3,5-dicarboxylic acid 5-methyl ester (IV) into 5,7-dihydro-2-methyl-4-(2-nitrophenyl)-5-oxofuro[3,4-b] pyridine-3-carboxylic acid methyl ester (V) by heating under acidic conditions, V was extracted with n-pentane-dichloromethane (7:3) and analysed on a C18 column with ultraviolet detection. Subsequently, 2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinedicarboxylic acid monomethyl ester (III) was extracted with chloroform and analysed on the same system. Limits of determination in blood were 0.1 microgram/ml for III and 0.05 microgram/ml for IV and V; these limits were two to ten times higher for urine. This inter-assay variability was always less than 7.5%.     

High-performance liquid chromatography assays for desmethoxyyangonin, methysticin, kavain and their microsomal metabolites

Three novel, simple and reproducible high-performance liquid chromatography quantitative assays with UV detection were developed and validated for three major kavalactones--desmethoxyyangonin, methysticin and kavain--in rat liver microsomes using diazepam as an internal standard; liquid-liquid extraction was used for sample preparation and analysis was performed on a Shimadzu 10A high-performance liquid chromatography system. The analysis was carried out in reversed-phase mode with a Luna C(18) column (150 x 2.00 mm, 3 microm) at 40 degrees C. The limit of quantitation was 0.1 microg/mL using 0.25 mL of microsomal solution. The assays were linear over the range 0.1-10 microg/mL for desmethoxyyangonin, methysticin and kavain. Quality control samples exhibited good accuracy and precision with relative standard deviations lower than 15% and recoveries between 85 and 105%. The assays exhibited satisfactory performance with high sensitivity for quantifying desmethoxyyangonin, methysticin and kavain in rat liver microsomes and were successfully used to determine the three kavalactones and their microsomal metabolites.     

High-performance liquid chromatography of gentian violet, its demethylated metabolites, leucogentian violet and methylene blue with electrochemical detection

High-performance liquid chromatographic conditions are reported for the electrochemical detection (ED) of Gentian Violet, its demethylated metabolites, Leucogentian Violet and Methylene Blue. Gentian Violet, its demethylated metabolites and Leucogentian Violet were separated within 14 min on a cyano column eluted isocratically with methanol-buffer (60:40) as the mobile phase. ED responses for Gentian Violet, Leucogentian Violet and Methylene Blue were linear over the ranges 0.54-6.75, 0.50-25.2, and 5.7-285 ng, respectively. Under these conditions, the compounds were eluted in the following order: Leucogentian Violet, N"-2-tetra-methylparaosaniline chloride, N'-1-tetramethylpararosaniline chloride, pentamethylpararosaniline chloride and Gentian Violet. Methylene Blue and Gentian Violet had essentially the same retention time under these parameters. The detection limit for Gentian Violet, its demethylated metabolites and Leucogentian Violet was determined to be 0.1 pmol. A detection limit of 3 pmol was established for Methylene Blue. Detector response, elution, separation, linearity and sensitivity of detection are discussed.     

High-performance liquid chromatography for the separation of angiotensin and its metabolites in human plasma and sweat

A reversed-phase high-performance liquid chromatography (HPLC) method with gradient elution for the separation of angiotensin peptides is described. The highly reproducible method allows the base-line separation of angiotensin peptides with UV detection at 225 nm. This chromatographic methodology in combination with radioimmunoassay (RIA) is used for the characterization of angiotensin peptides in human plasma and sweat.     

High-performance liquid chromatographic separation of cadralazine from its potential metabolites and degradation products. Quantitation of the drug in human plasma and urine

The chromatographic behaviour of cadralazine and its potential metabolites and degradation products with respect to pH, buffer molarity and composition of eluent is described. A selective method with an adequate sensitivity for the determination of the drug in human plasma and urine is also reported. The method includes extraction of biological fluids with chloroform and the analysis of extracts on a reversed-phase column with isocratic elution and detection at 254 nm. The method has been applied to the analysis of plasma and urine of a patient administered a single oral dose of 30 mg of cadralazine.     

High-performance liquid chromatography systems for the separation of benzodiazepines and their metabolites

The high-performance liquid chromatographic (HPLC) retention characteristics of 21 benzodiazepine drugs and some of their metabolites have been examined on both silica and ODS-silica packing materials. Four HPLC systems have been considered and retention data are presented for the drugs on these systems. The correlation of retention data on the systems is considered with reference to the problem of identifying unknown benzodiazepines.     

High-performance liquid chromatographic determination of pilocarpine hydrochloride and its degradation products using a beta-cyclodextrin column.

A high-performance liquid chromatographic (HPLC) method for the determination of pilocarpine, isopilocarpine, pilocarpic acid and isopilocarpic acid was developed. A beta-cyclodextrin column achieved the separation in less than 10 min. Baseline resolution of all four compounds permitted the determination of each degradation product in the presence of pilocarpine. The calibration graphs for each compound were linear, and pilocarpine degradation products could be determined without a correction for the ultraviolet detector response using a pilocarpine standard. A comparison of beta-cyclodextrin separation with the USP HPLC method demonstrated similar results for pilocarpine contents in several commercial ophthalmic formulations.     

High-performance liquid chromatography method for simultaneous determination of aliphatic acid, aromatic acid and neutral degradation products in biomass pretreatment hydrolysates

A variety of degradation products are produced upon dilute acid pretreatment of lignocellulosic biomass. Within this larger construct, organic acids, phenols and aromatic aldehydes represent important compound classes to investigate due to increasing evidence of their inhibitory effect on fermentative microorganisms. An analytical extraction procedure is presented, enabling isolation of potential analytes away from alternative products in biomass hydrolysates. Additionally, a reversed-phase high-performance liquid chromatography (HPLC) method has been developed and validated, affording simultaneous separation and quantitative determination of 32 potential analytes in water with UV detection at 210 nm. The method was subsequently employed to quantify a variety of aliphatic acid, aromatic acid, aldehyde and phenolic degradation products in a corn-stover hydrolysate at concentration levels ranging from 0.02 to 41 mM.