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 wave-length 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.     

Determination of luxabendazole in biological fluids by high-performance liquid chromatography.

Luxabendazole, a new benzimidazole, is a highly potent broad-spectrum anthelmintic. A high-performance liquid chromatographic method has been developed for its determination in serum and urine samples. In order to optimize the clean-up of samples we compared two procedures: C18 Sep-Pak cartridges and ultrafiltration through a cellulose membrane with a 30,000 relative molecular mass cut-off. In order to obtain the most suitable mobile phase, we studied the influence of pH and acetonitrile content on the capacity factor (k'). Chromatographic separation and quantification were performed on a reversed-phase column packed with 5-microns Nucleosil C18. The mobile phase was acetonitrile-0.05 M phosphate buffer (pH 7.0), (40:60, v/v). The column effluent was monitored by ultraviolet-visible spectrophotometry at 290 nm. The method shows good recovery, precision and accuracy. The lower limit of detection for luxabendazole is 15 ng/ml in serum samples and 25 ng/ml in urine samples.     

Determination of tertatolol enantiomers in biological fluids by high-performance liquid chromatography.

A stereospecific high-performance liquid chromatographic method for the quantification of (-)- and (+)-tertatolol in plasma and urine is described. The method involves solid-phase extraction followed by derivatization with S(+)-naphthylethylisocyanate to form the urea derivative, which is more sensitive to fluorescence detection. The separation of the diastereomeric derivatives was performed by reversed-phase high-performance liquid chromatography. Fluorimetric detection (lambda excitation = 220 nm, lambda emission = 320 nm) allows the quantification of tertatolol enantiomers down to 6 ng/ml. The assay was used to study the pharmacokinetic profile of tertatolol enantiomers following oral administration of racemic tertatolol; preliminary results suggest enantioselective absorption and/or disposition of tertatolol.     

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 normalphase (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, providng nearly quantitative extraction (greater than 95%) and a sensitivity limit of 0.01 to 0.02 microgram/ml blood or plasma, without interference from sulfonamides.     

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

A rapid, selective and sensitive assay has been developed for the determination of the anti-anginal drug, bepridil, in biological samples. The lowest concentration of bepridil which can be measured accurately and precisely in a 2-ml plasma or urine sample is 10 ng/ml. The standard curve is linear in the concentration range 10-2000 ng/ml. Accuracy and precision of the assay, expressed as relative deviation and coefficient of variation (inter-run) are less than 6.5% at all concentrations in the linear range. No interfering peaks are observed. Using an automatic injector and a laboratory computer system, 48 samples are analyzed routinely in an 8-h day.     

Ion-exchange high-performance liquid chromatography in the brewing industry

Isocratic HPLC methods using an ion-exchange column (Aminex HPX) for the determination of carbohydrates, organic acids, alcohols (methanol, ethanol, glycerol), 5-hydroxymethylfurfural and purines in wort, beer, wine and soft drinks are presented. The strategy for selecting the best column geometry, the appropriate counter ion and specific detection systems is discussed.     

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, delta 4-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.     

A rapid and specific high-performance liquid chromatographic assay for theophylline in biological fluids

A rapid, specific high-performance liquid chromatographic analysis of theophylline in plasma, serum, and saliva is described. Proteins present in the biological samples are precipitated with 6% perchloric acid and the clear supernatant is chromatographed on a reversed-phase column. Only 100 microL of serum is required and concentrations as low as 0.07 micrograms/mL can be measured accurately. Other xanthines do not interfere in the assay. Within- and between-day variation is less than or equal to 2.2%. The method shows less bias and greater precision than the TDx (Abbott Diagnostics) procedure commonly used in clinical laboratories.