Determination of trimethoprim in biological fluids by high-performance liquid chromatography

A rapid, sensitive, and specific high-performance liquid chromatographic assay was developed for the determination of trimethoprim in blood, plasma, and urine using normal-phase (adsorption) chromatography on a microparticulate silica column and UV monitoring at 280 nm. Trimethoprim is selectively extracted from the biological sample matrix at alkaline pH with chloroform, providing nearly quantitative extraction (>95%) and a sensitivity limit of 0.01 to 0.02 μg/ml blood or plasma, without interference from sulfonamides.     

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.     

Determination of alpha-amanitin and beta-amanitin in human biological fluids by high-performance liquid chromatography

A high-performance liquid chromatographic assay of alpha-amanitin and beta-amanitin in human serum, urine, or stomach washings is described. Sample preparation involves a chemical step with deproteinization and organic solvent treatment, and a selective cleanup and concentration step on reversed-phase prepacked cartridges. Separations are performed on a reversed-phase analytical column under isocratic conditions with uv detection at 280 nm. The method allows the quantitation of alpha- and beta-amanitin separately with a detection limit of 10 ng/ml for both toxins.     

Analysis of isoxazolyl penicillins and their metabolites in body fluids by high-performance liquid chromatography

A high-performance liquid chromatographic assay method to quantitate the isoxazolyl penicillins, their active metabolites, and their penicilloic acids in serum or urine is described. Separation and analysis is performed using reversed-phase chromatography. Urine samples, after the appropriate dilution, can be assayed directly. Serum samples (0.1 ml) are either extracted with methylene chloride or treated with perchloric acid—methanol. Serum levels as low as 0.4 μg/ml (extraction procedure) can be assayed accurately.     

Determination of cholesterol in sub-nanomolar quantities in biological fluids by high-performance liquid chromatography

A highly sensitive method for the determination of cholesterol in biological fluids is described. Unsaponifiable lipids from rat serum and thoracic duct lymph chylomicron samples were treated with cholesterol oxidase. The product of the enzymatic reaction, Δ⁴-cholestenone, was analysed by normal-phase high-performance liquid chromatography (HPLC) using hexane—isopropanol (95:5, v/v) as a mobile phase and detected with a UV spectrophotometer at 240 nm. When the standard samples containing varying amounts of cholesterol (0.15–3 nmol) were treated with cholesterol oxidase and analysed by HPLC (injected amounts 0.09–1.8 nmol of cholesterol), the peak areas increased proportionally with the amounts of authentic cholesterol with a correlation coefficient of 0.996. The values in these biological fluids determined by the HPLC method were identical to those obtained by enzymatic—colorimetric or gas chromatographic methods. Moreover, the detection limit (0.09 nmol) of the present method (0.15 nmol are required for the sample preparation) is lower than those of conventional methods (approximately 30 nmol). Because of the excellent sensitivity and reproducibility, this method is well suited for the determination of cholesterol in biological fluids where cholesterol concentration is low.     

Quantitative analysis of sulpiride in body fluids by high-performance liquid chromatography with fluorescence detection

A high-performance liquid chromatographic method for the analysis of sulpiride, N-ethyl-2-(2-methoxy-5-sulphonamido-benzamido-methyl)-pyrrolidine, in body fluids is described. A structurally related compound, N-ethyl-2-(2,4-dimethoxy-benzamido-methyl)-pyrrolidine, was used as internal standard.A fluorescence detector with excitation maximum at 299 nm and emission maximum at 342 nm was used for the quantitation. The detection limit was about 10 ng/ml in serum and cerebrospinal fluid and about 200 ng/ml in urine. The experimental error was 5–10% in the concentration range 25–100 ng/ml. Some preliminary data from a pharmacokinetic study in healthy volunteers are presented. The half-life for sulpiride in serum was about 8 h. Sulpiride was also measured in cerebrospinal fluid from five drug-treated psychotic patients.     

Corticosteroid analysis in biological fluids by high-performance liquid chromatography

A sensitive, specific, and reproducible high-performance liquid chromatographic assay for the simultaneous determination of prednisone, prednisolone and cortisol in biological fluids was developed with dexamethasone as the internal standard. Samples are extracted with methylene chloride, washed with sodium hydroxide and then water, and chromatographed on a microparticulate silica gel column with UV detection at 254 nm. Sensitivity was greater than 15 ng for all four steroids. Specificity was supported by use of dual wavelength UV detection and/or radioimmunoassay. The assay has been applied in pharmacokinetic studies and a typical plasma concentration—time profile for the three steroids is presented for one subject who received 50 mg of prednisone.     

Laccase assay by means of high-performance liquid chromatography

Spectrophotometric determination of laccase activity may be affected by the formation of quinoid chromophores arising from nonenzymatic oxidations interfering with enzymatic reactions. Km values for guaiacol obtained by spectrophotometric and HPLC methods confirm the above hypothesis. HPLC results are particularly useful for the assay of laccase activity on natural phenolic extracts.     

Determination of trimethoprim in bovine serum by high-performance liquid chromatography with confirmation by thermospray liquid chromatography—mass spectrometry

A high-performance liquid chromatographic (HPLC) method with a detection limit of 5 ng/ml was developed for the analysis of trimethoprim in bovine serum. Trimethoprim and the internal standard, ormetoprim, under alkaline conditions, were first extracted into dichloromethane and then back-extracted into dilute sulphuric acid (0.15 M) and cleaned-up on a C₁₈ cartridge. Trimethoprim was quantified on a C₁₈ column using a triethylammonium acetate—acetonitrile—methanol (16:3:1, v/v/v) mobile phase at a flow-rate of 1.5 ml/min, with ultraviolet detection at 225 nm. This method was used to verify the accuracy of test responses obtained with the Brilliant Black Reduction test, a rapid screening method, for trimethoprim levels in the serum of steers treated with Trivetrin. Confirmation of the presence of trimethoprim in the sample extract was obtained by thermospray HPLC—mass spectrometry.     

Determination of cystamine by high-performance liquid chromatography

A highly sensitive and specific assay method for cystamine using high-performance liquid chromatography has been developed. The method is based on postcolumn derivatization of cystamine with o-phthaladehyde in the presence of 2-mercaptoethanol and sodium hypochlorite. The separation of cystamine was achieved using a cation exchange column (ISC-05/S0504). The assay was linear over the concentration range of 2 to 200 pmol. For the application of this assay method to biological materials, the pretreatment with a cation exchange column (Dowex 50W X 8) was necessary to remove interfering o-phthaladehyde-reactive substances. Since cysteamine in biological materials was quantitatively converted to cystamine during these sampling procedures, this method was found to be suitable for assaying the cysteamine plus cystamine content in various organs and tissues. The cysteamine-cystamine content in various tissues of rat determined by the present assay method has been presented.