Ion-pair high-performance liquid chromatographic determination of isoniazid and acetylisoniazid in plasma and urine. Application for acetylator phenotyping

Isotachophoresis seems a likely candidate for a reference method, as it is more accurate and precise than common routine methods. The response is highly linear and depends on a well defined transport number of the leading ion, stability of the driving current, mobility of the separand, which is well controlled by the leading electrolyte and the use of a high-resolution detector. The suitability of isotachophoresis as a reference method was investigated for the determination of sodium in human serum. The operational conditions were 0.01 M K+/citrate (leading electrolyte) at pH 5.5 and 0.01 M creatinine X HCl (terminating electrolyte). Both n-butylamine and ammediol could be used as internal standards. The calibration graph constructed from standard solutions, diluted by weight with the internal standard, yielded a correlation coefficient of 0.99994 (n = 50) in the working range. The method seems especially useful for the determination of any ionic solute in, e.g., clinical samples (lithium, calcium, creatinine or drugs).     

Simple high-performance liquid chromatographic method for the determination of tolbutamide and its metabolites in human plasma and urine using photodiode-array detection

A high-performance liquid chromatographic method has been developed for the determination of tolbutamide and its metabolites in human plasma and urine. The compounds examined were extracted with diethyl ether from the acidified biological fluid. Chlorpropamide was used as internal standard, and 235 nm was chosen as the wavelength for diode-array detection. A study of the relationship between the capacity factor and the mobile phase composition and pH showed that acetonitrile-2-propanol-0.1% orthophosphoric acid (17: 17: 66, v/v) was the best eluent on a C8 reversed-phase column. The method is precise, sensitive and suitable for pharmacological investigations.     

Automated high-performance liquid chromatographic determination of antipyrine and its main metabolites in plasma, saliva and urine, including 4,4'-dihydroxyantipyrine

A rapid, selective and sensitive method was developed for the determination of antipyrine and its main metabolites in plasma, saliva and urine by an automated high-performance liquid chromatographic system. Using a MOS-Hypersil reversed-phase column with a phosphate buffer--acetonitrile mobile phase, baseline separation of antipyrine, its metabolites 3-hydroxymethylantipyrine, norantipyrine and 4-hydroxyantipyrine, and the internal standard, phenacetin, was achieved within 6 min. Factors regarding the accuracy and precision of the method and the stability of phase I metabolites during sample preparation are discussed, taking into account certain drawbacks of previously published methods. Based on the same chromatographic system a method was developed for the assay of 4,4'-dihydroxyantipyrine in urine. This compound is an important metabolite of antipyrine in the rat, representing 12.6 +/- 1.8% of the administered dose (n = 18).     

High-performance liquid chromatographic determination of nicotinamide and its metabolites in human and murine plasma and urine.

A high-performance liquid chromatographic method is described which enables the determination of nicotinamide and eight of its possible metabolites in human and murine plasma and urine, using ion-pairing on a base-deactivated reversed-phase column. Calibration curves were linear up to 2 mumol/ml for nicotinamide and 200 nmol/ml for the metabolites; both the intra- and inter-assay relative standard deviations ranged between 1 and 8%. In murine plasma, the N-oxide was the major nicotinamide metabolite, but in man, formation of 1-methylnicotinamide and the 2- and 4-pyridones was also significant. In urine, nicotinuric acid was seen in the mouse, but no nicotinic acid metabolites were seen in man.     

Improved high-performance liquid chromatographic determination of ciprofloxacin and its metabolites in human specimens.

A simple approach to the quantitation of ciprofloxacin and its three metabolites, M1 (desethylene-ciprofloxacin), M2 (sulfo-ciprofloxacin) and M3 (oxo-ciprofloxacin), in human serum, urine, saliva and sputum is described. This assay allows the parent drug and its metabolites to elute and be resolved in a single chromatogram at 280 nm using a linear gradient. The procedure involved liquid-liquid extraction. Separation was achieved on a C18 reversed-phase column. The limit of detection of ciprofloxacin is 0.05 microgram/ml and that of its three metabolites is 0.25 microgram/ml. This method is sufficiently sensitive for pharmacokinetic studies.     

High-performance liquid chromatographic determination of flurazepam and its metabolites in human blood plasma

A blood plasma sample was spiked with flurazepam and its metabolites and diluted with water (1 + 1); after protein precipitation, flurazepam and three of its metabolites were extracted into ethyl acetate at pH 9. After evaporation of the solvent, the residue was dissolved in the mobile phase for injection onto a reverse-phase LiChrosorb column; eluents were methanol-water (62:38) for separation of the metabolites and methanol-phosphate buffer (85:15) for flurazepam. The limit of detection varied from 0.1 to 1.6 ng. Recoveries from spiked plasma (1 μg ml^?1) were 94–95%. At 254 nm, the response was usually linear over the range 5–50 ng.     

Simple high-performance liquid chromatographic method for the determination of a new phytochemical drug, fellavine, and its metabolites in human and rat plasma and urine.

The use of a small precolumn instead of an injection loop for the determination of a new phytochemical drug, fellavine, and its metabolites is described. The method combines the direct injection of plasma and urine into the reversed-phase precolumn with separation on a Spheri-5 RP-18 analytical column. Different sorbents in the precolumn were compared. A recovery of fellavine and its metabolites from biological fluids except rat plasma of almost 100% was achieved on Chrompack RP (30-40 microns) and LiChrosorb RP-18 (7 microns). For rat plasma only the last sorbent gave 80% fellavine recovery. The influence of the protein binding on the fellavine recovery was examined. The limit of detection was equal to 0.05 micrograms/ml fellavine for plasma and 0.02 micrograms/ml for urine. To enhance the limit of detection longer precolumns were perferred.     

Improved high-performance liquid chromatographic method for the simultaneous determination of caffeine and its N-demethylated metabolites in plasma using solid-phase extraction

An improved high-performance liquid chromatographic method for the simultaneous determination of caffeine and its N-demethylated metabolites in plasma is described. Excellent resolution of all components is provided by reversed-phase chromatography using a mobile phase consisting of 1% acetic acid-methanol (83:17) at a flow-rate of 2.7 ml/min, in conjunction with a Waters Assoc. Nova-Pak C18 column which was protected by a Waters Assoc. Guard-Pak precolumn module containing a Guard-Pak CN cartridge. Rapid extraction of caffeine and the dimethylxanthines from plasma was achieved using reversed-phase octadecylsilane bonded-silica columns (Bond-Elut C18). With only 100 microliters of sample, plasma levels in the region of 50 ng/ml for the dimethylxanthines and 100 ng/ml for caffeine can be determined using ultraviolet detection at 273 nm. The method has been used for measuring umbilical cord plasma samples to provide information regarding foetal exposure to caffeine and its metabolites and is also suitable for therapeutic drug monitoring of caffeine and theophylline levels in the treatment of neonatal apnoea.     

High-performance liquid chromatographic determination of lansoprazole and its metabolites in human serum and urine.

A simple and sensitive high-performance liquid chromatographic method with ultraviolet detection is described for the simultaneous determination of lansoprazole and its metabolites in human serum and urine. The analytes in serum or urine were extracted with diethyl ether-dichloromethane (7:3, v/v) followed by evaporation, dissolution and injection into a reversed-phase column. The recoveries of authentic analytes added to serum at 0.05-2 micrograms/ml or to urine at 1-20 micrograms/ml were greater than 88%, with the coefficients of variation less than 7.1%. The minimum determinable concentrations of all analytes were 5 ng/ml in serum and 50 ng/ml in urine. The method was successfully applied to a pharmacokinetic study of lansoprazole in human.     

Stereospecific high-performance liquid chromatographic assay of acebutolol in human plasma and urine

A sensitive high-performance liquid chromatographic technique is described for the separation of R- and S-acebutolol in human plasma and urine. The procedure involves derivatization with the chiral reagent S-(+)-1-(1-naphthyl)ethyl isocyanate. The resulting diastereoisomers are quantified using normal-phase high-performance liquid chromatography with fluorescence detection (220/389 nm). Virtual baseline separation, free from interference, with achieved (resolution factor = 1.45). Excellent linearity (r greater than 0.998) was observed throughout the range 10-500 ng/l and 2-100 mg/l in plasma and urine, respectively. Inter-assay variability was less than 5% for each enantiomer at concentrations of 10 ng/ml. This method is applicable for the determination of the pharmacokinetics, in man, of acebutolol enantiomers in plasma and urine.