High-performance liquid chromatographic determination of rufloxacin and its main active metabolite in biological fluids.

A high-performance liquid chromatographic method is described for the determination of a fluoroquinolone, rufloxacin, and its N-desmethyl metabolite in plasma, urine and bile. Samples are chromatographed on a poly(styrene-divinylbenzene) column, the eluate being monitored with a fluorescence detector. The method was validated and a detection limit of 10 ng/ml for both rufloxacin and its metabolite in all the biological matrices considered was found. The method was successfully applied in pharmacokinetic studies.     

High-performance liquid chromatographic determination of diclofenac and its monohydroxylated metabolites in biological fluids

Sensitive and selective high-performance liquid chromatographic assays for diclofenac and its monohydroxylated metabolites in biological fluids are described. Using ultraviolet detection at 282 nm, diclofenac is assayed in plasma at concentrations down to 10 ng/ml; total (free + conjugated) diclofenac and its monohydroxylated metabolites (the sum of 3'- + 4'-hydroxydiclofenac and 5-hydroxydiclofenac) are assayed in urine after chemical hydrolysis at concentrations down to 200 ng/ml. The applicability of the described assays is shown.     

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 determination of ibuprofen, its metabolites and enantiomers in biological fluids

An isocratic high-performance liquid chromatographic method to determine racemic ibuprofen (assay I) and its major metabolites (assay II) in biological fluids (plasma, urine, bile) using a conventional reversed-phase column is described. A third assay using beta-cyclodextrin as stationary phase (Cyclobond I) for the separation of the ibuprofen enantiomers is also described. A wavelength of 220 nm was used to monitor the substances. The sensitivity of the method was 0.1 microgram/ml for all three assays. The method was demonstrated to be suitable for stereoselective pharmacokinetic studies of ibuprofen in humans and animals.     

High-performance liquid chromatographic methods for the determination of ranitidine and its metabolites in biological fluids

Summary The use of an existing reversed-phase ion-pair method for the determination of ranitidine, ranitidine-N-oxide, ranitidine-S-oxide and desmethylranitidine in the plasma of patients taking the anti-ulcer drug, ranitidine is described. The development of a ternary reversed-phase system which is more suitable for the routine determination of ranitidine and the three metabolites is reported. This system has been used to determine quantitatively ranitidine and the metabolites in urine. Studies in animals using¹⁴C-ranitidine have shown that ranitidine is also oxidatively deaminated to a 5-substituted, 2-furan carboxylic acid. A reversed-phase ion-pair system, in which cetrimide is the counter ion, has been developed for the quantitative determination of the 5-substituted, 2-furan carboxylic acid and ranitidine-N-oxide in urine and faeces from patients treated with ranitidine. Presented at the 15th International Symposium on Chromatography, Nürnberg, October 1984     

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 chromatographic determination of spironolactone and its metabolites in human biological fluids after solid-phase extraction.

A simple and sensitive high-performance liquid chromatographic procedure to determine spironolactone and its three major metabolites in biological specimens is described. The assay involves sequential extraction on C18 and CN solid phases, and subsequent separation on a reversed-phase column. In plasma samples, spironolactone and its metabolites were completely separated within 8 min using an isocratic mobile phase, while in urine samples a methanol gradient was necessary to achieve a good separation within 14 min. Recoveries for all analytes were greater than 80% in plasma and 72% in urine. Linear responses were observed for all compounds in the range 6.25-400 ng/ml for plasma and 31.25-2000 ng/ml for urine. The plasma and urine methods were precise (coefficient of variation from 0.8 to 12.5%) and accurate (-12.1% to 7.4% of the nominal values) for all compounds. The assay proved to be suitable for the pharmacokinetic study of spironolactone in healthy human subjects.     

High-performance liquid chromatographic evaluation of 2-(alpha-thenoylthio)propionylglycine and its two metabolites in biological fluids

A high-performance liquid chromatographic (HPLC) method for determining 2-(alpha-thenoylthio)propionylglycine (TTPG) and its two main metabolites, thiophenecarboxylic acid and thiopronine, in biological samples was developed. TTPG and its metabolites were extracted by solvent partition and then determined by reversed-phase HPLC with UV detection at 245, 295 and 360 nm. This procedure was validated in order to allow the assay of these compounds in plasma and urine samples with sufficiently low detection limits (50 ng/ml for TTPG and TCA and 100 ng/ml for thiopronine) and with good linearity within the concentration range investigated. It was applied to a comprehensive pharmacokinetic investigation of TTPG in healthy volunteers.     

Determination of tannic acid and its phenolic metabolites in biological fluids by high-performance liquid chromatography.

A method for the identification and determination of tannic acid and its phenolic metabolites in biological fluids by high-performance liquid chromatography was developed. Tannic acid and four phenolic compounds, namely gallic acid, pyrogallol, 4-O-methylgallic acid and ellagic acid, were successfully extracted from the biological fluids by using ethyl acetate at acidic conditions. Gallic acid, pyrogallol and 4-O-methylgallic acid were found in the sheep urine, gallic acid, 4-O-methylgallic acid and ellagic acid in plasma, and gallic acid and ellagic acid in abomasal fluid after abomasal dosing of tannic acid. Tannic acid was found in the plasma apart from the abomasal fluid into which it was administered. The concentrations of tannic acid, gallic acid, pyrogallol, 4-O-methylgallic acid and ellagic acid in plasma, abomasal fluid and urine were measured. This method could be applied to measurement of other hydrolysable tannins and their phenolic metabolites in biological materials.     

Determination of disulfiram and metabolites from biological fluids by high-performance liquid chromatography

A high-performance liquid chromatographic method is described for the determination of disulfiram, diethyldithiocarbamate, diethyldithiocarbamate methyl ester, carbon disulfide, and diethylamine from a single sample of plasma or urine. The analytical procedure is based on a quantitative stepwise extraction of disulfiram and diethyldithiocarbamate methyl ester, or the conversion of diethyldithiocarbamic acid, carbon disulfide and diethylamine to diethyldithiocarbamate methyl ester for chromatographical determination. The procedure is specific, precise and simple. The application of the analytical methods developed for the determination of disulfiram and the various metabolites in plasma from mice given disulfiram intraperitoneally or humans given Antabuse orally is illustrated.     

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.     

An assay of sulphasalazine,sulphapyridine and its acetylated and hydroxylated metabolites in biological fluids by liquid chromatography

Summary An HPLC assay was developed for the determination of sulphasalazine (salicylazosulphapyridine), and its main metabolites; sulphapyridine, acetylsulphapyridine, hydroxysulphapyridine and acetylhydroxysulphapyridine in human serum, synovial fluid and urine. Sep-Pak C₁₈ cartridge was used for the extraction of the investigated compounds and the added sulphadimidine internal standard. The methylene chloride-chloroform-methanol eluate was evaporated and the dissolved dry residue was injected onto a Hypersil ODS (100×2.1 mm) 5 m microbore, analytical column. A linear gradient was used, the mobile phase was ternary. The assay is sufficiently sensitive for routine therapeutic drug monitoring and phenotyping in patients treated with sulphasalazine.     

Quantitation of chlordiazepoxide and its metabolites in biological fluids by thin-layer chromatography.

Chlordiazepoxide and its 4 major metabolites were assayed after separation by thin-layer chromatography following extraction from biological fluids. The compounds become intensely fluorescent in the presence of red, fuming nitric acid. The resulting compounds are quantitated with a spectrodensitometer with a fluorescent attachment. The sensitivity varies between 0.05 and 0.1 microgram. The coefficient of variation is 1.4% for assays in urine and 6.4% in serum.     

High-performance liquid chromatographic analysis of the mycotoxin citrinin and its application to biological fluids

Critinin is a toxic metabolite produced by several species of Penicillium and Aspergillus. Citrinin is nephrotoxic and has been implicated in disease outbreaks in animals and humans. Citrinin was resolved as a sharp peak by reversed-phase high-performance liquid chormatography on a small-particle 10μm) column by elution in 4.25 min with a phosphoric acid (0.25 nN)-acetonitrile-2-propanol solvent (55:35:10). Detection was by ultraviolet absorbance at 340 nm. The relationship between peak height and area and quantity injected was linear over a range of 2–50 ng at 340 mn and 5–200 ng at 365 nm. Retention time and peak area were highly reproducible. As little as 2–5 ng citrinin was detectable. Complete recovery of citrinin from plasma samples containing known quantities of [¹⁴C]citrinin was obtained over a range of 5–40 μg/ml by treatment of the plasma with 1 N hydrochloric acid followed by extraction with ethyl acetate. The method provides for the direct analysis of citrinin in urine and bile without prior extraction.     

High-performance liquid chromatographic determination of loxoprofen and its diastereomeric alcohol metabolites in biological fluids by fluorescence labelling with 4-bromomethyl-6,7-methylenedioxycoumarin

A simple and sensitive high-performance liquid chromatographic procedure to determine loxoprofen and its diastereomeric alcohol metabolites in biological specimens is described. The analysis involves liquid-liquid extraction with benzene, pre-column derivatization with a highly fluorogenic reagent, 4-bromomethyl-6,7-methylenedioxycoumarin (BrMDC) and subsequent separation on a reversed-phase column. Loxoprofen, its pharmacologically active metabolite, trans-alcohol, and less active cis-alcohol were completely separated within 20 min with a mobile phase of 55% of aqueous acetonitrile containing acetic acid. Any endogenous substances do not interfere in the analysis of either plasma or urine samples. The quantitation limit was 0.01 micrograms/ml for human plasma and 0.05 micrograms/ml for urine. The method was applied to a pharmacokinetic study in healthy human subjects who had received 60 mg of loxoprofen sodium.