High-pressure liquid chromatographic determination of thioridazine and its major metabolites in biological tissues and fluids

A method is described for the determination of thioridazine and its major metabolites in postmortem tissues and fluids by enzymic digestion, followed by a micro-extraction technique using ethyl acetate. Quantitation was performed by using a cyano-bonded silica (mu Bondapak-CN) column packing and a mobile phase consisting of n-octylamine, methanol, and water.     

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-pressure liquid chromatographic determination of some 1,4-benzodiazepines and their metabolites in biological fluids: a review

In recent years the need for rapid, sensitive and specific assays for benzodiazepines has resulted in the publication of a number of high-pressure liquid chromatographic (HPLC) methods for their determination. This paper reviews the methods available to date for the determination of chlordiazepoxide, clonazepam, diazepam, flurazepam, lorazepam, nitrazepam, oxazepam and their metabolites in biological fluids.     

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 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 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 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 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.     

Versatile isocratic high-performance liquid chromatographic assay for propranolol and its basic, neutral and acidic metabolites in biological fluids

A comprehensive high-performance liquid chromatographic method was developed for quantitating propranolol and its known metabolites in serum, bile and urine. Analysis was performed before and after incubation of the samples with beta-glucuronidase-arylsulfatase to quantitate both free and conjugate forms of the oxidative metabolites. Fractionation of the basic, neutral and acidic metabolites was achieved by differential pH solvent extraction. The basic and neutral metabolites were extracted from the biological samples at pH 10.5 with 2% n-butanol in dichloromethane. Additional clean-up of the basic fraction by back-extraction into dilute acid was needed for those samples that were subjected to enzymatic hydrolysis. The original aqueous sample was titrated with acid to pH 1, followed by extraction of the remaining acidic metabolites into either n-butanol-dichloromethane (with unhydrolyzed serum) or carbon tetrachloride (with all other samples). Chromatographic separation of the metabolites in the different extracts was achieved on a reversed-phase C18 column, using a single isocratic mobile phase consisting of 0.044 M pH 2.7 phosphate buffer, tetrahydrofuran, methanol and acetonitrile, with the addition of n-butylamine as a competing base to control retention volume and peak shape. Detection and quantitation of propranolol and its metabolites in the low nanogram to sub-nanogram range was afforded by fluorescence at a low UV excitation wavelength. The coefficients of variation for replicate assay of spiked samples were uniformly less than 6% for all the analytes.     

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.     

Improved high-performance liquid chromatographic assay for encainide and its metabolites in human body fluids

Methods reported previously for the determination of encainide and its metabolites in biological fluids have not been extensively described and evaluated. We report an improved high-performance liquid chromatographic assay for the quantification of these compounds in plasma and urine with complete estimation of the accuracy and reproducibility of the analytical method. The major improvements consist of: (1) the use of ethaverine as an appropriate internal standard; (2) the use of the salting-out technique which improves the extraction recovery for the metabolites of encainide and the sensitivity of the assay; and (3) a shift of the ultraviolet absorption wavelength from 254 to 270 nm to increase the selectivity of the detection.     

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.     

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.     

High-pressure liquid chromatographic analysis of carbofuran and two non-conjugated metabolites in crops as fluorescent dansyl derivatives

Carbofuran, and non-conjugated 3-hydroxycarbofuran and 3-ketocarbofuran were extracted from carrots, corn and potatoes with acetone and partitioned into hexane-methylene chloride. The organic extract was evaporated to a small volume for clean-up on a 2% deactivated Florisil column. All three carbamates were eluted with 15% acetone in hexane. The pesticide residues were hydrolysed to their corresponding phenols with 0.1 M sodium carbonate followed by derivatization with dansyl chloride in acetone. The derivatives were extracted and analysed by high-pressure liquid chromatography with fluorescence detection (excitation, 360 nm; emission, greater than 400 nm). Absolute recoveries for all three compounds were between 50 and 65% for spiked samples by the extraction method used. Detection limits approached 0.01 ppm in the foods studied.