Analysis of diclofenac and four of its metabolites in human urine by HPLC.

An HPLC method for the determination of diclofenac (DCF) and four of its metabolites (3'-hydroxydiclofenac, 4'-hydroxydiclofenac, 5-hydroxydiclofenac, and 3'-hydroxy-4'-methoxydiclofenac) in human urine is described. Following base hydrolysis, the samples were neutralized and extracted. Evaporated extracts were reconstituted in mobile phase containing ascorbic acid, and chromatographed, using flow-rate programming, on a reversed-phase column. Absolute recovery (average), was at least 78% for diclofenac and ranged from 75 to 85% for the four metabolites. Standard curves showed linearity over the range of concentrations of 0.2 to 40 ug/mL, using 0.25 mL of urine. Specificity was demonstrated by examining chromatograms of extracts of blank urine from 8 volunteers and 24 study subjects. Good accuracy was observed for all compounds over the concentration range of 0.2 to 40 ug/mL using 0.25 mL of urine. Based on accuracy and precision criteria, the limit of quantitation for all 5 analytes was 0.4 ug/mL, using 0.25 mL of urine. Analysis of urine from subjects with normal and reduced renal function who received diclofenac orally demonstrated that total diclofenac and metabolites excreted in the urine represented approximately 31% and 4% of an oral dose of diclofenac, respectively.

Circadian rhythms in theophylline disposition: simulations and observations in the dog.

A constant-rate iv infusion of aminophylline approaching steady state in four dogs was used to investigate a circadian rhythm in theophylline disposition. A significant circadian rhythm (p less than 0.05, group cosinor analysis) was observed for serum theophylline concentrations and for the urinary excretion rates of theophylline and 3-methylxanthine. The phase relationship of the observed circadian rhythms in urinary pH to circadian changes in serum theophylline concentrations were supportive of a circadian rhythm in renal clearance of theophylline. Computer simulations indicated that circadian changes in serum theophylline concentration and urinary excretion of theophylline and its metabolites could be accounted for by a circadian rhythm in theophylline renal clearance and metabolism to 3-methylxanthine. A circadian rhythm in theophylline volume of distribution could not be totally dismissed as potentially having some effect on disposition. The timing of food intake is hypothesized to play a role in synchronizing the observed rhythm in serum theophylline concentrations.

An evaluation of ibuprofen bioinversion by simulation.

Using a pharmacokinetic model recently proposed to explain ibuprofen disposition in man, plasma concentrations of pure ibuprofen enantiomers were simulated following oral administration of (-)-(R)-ibuprofen, (+)-(S)-ibuprofen, or rac-ibuprofen. Simulated and literature values for AUC's were used to compare S/R ratios for different cases of the model and for different methods of calculating the fraction of R bioinverted to S. Numerical simulation using STELLA confirmed previous results for different cases of bioinversion. Simulated S/R AUC ratios, for administration of the racemate, ranged from 4.0 (presystemic bioinversion) to 1.66 (systemic bioinversion). Literature values for S/R AUC ratios averaged 1.53 +/- 0.2 for administration of the racemate; therefore, systemic bioinversion was concluded to be representative of ibuprofen disposition. Additional simulations of S/R AUC ratios, for administration of (-)-(R)-ibuprofen only, ranged from 1.5 (presystemic bioinversion) to 0.66 (systemic bioinversion). Literature values for S/R AUC ratios averaged 0.50 +/- 0.9 for administration of (-)-(R)-ibuprofen only, which again supported conclusions of systemic bioinversion. Using different equations for estimation of fraction of R inverted to S (FR----S), results based on simulated data were identical; however, FR----S values based on literature data were different. Therefore, assumptions made for different FR----S equations do not appear to be rigorous. Calculations of FR----S, based on literature data, averaged 0.52 overall, indicating bioavailability of (+)-(S)-ibuprofen may be similar for a 150 mg dose of (+)-(S)-ibuprofen compared to a 200 mg dose of racemate.

Circadian rhythm in theophylline disposition during a constant-rate intravenous infusion of aminophylline in the dog.

The disposition of theophylline in three dogs was determined during a 48-h constant-rate intravenous infusion of aminophylline. A systematic fluctuation in serum theophylline concentrations was observed over a 24-h period, which appeared to be characteristic of a circadian rhythm. Neither assay variability nor fluctuations in the infusion pump rate could account for the observed variations in the serum concentrations. It was concluded that the changes in the theophylline concentrations were the result of a circadian rhythm in theophylline disposition.

A new substrate for the measurement of N-acetyltransferase activity.

A sensitive colorimetric method for the measurement of N-acetyltransferase (NAT) is described. It is based on the high rate of acetylation of 2-(p-aminobenzamido)pyridine by the liver enzyme and the lack of it by the blood NAT. A linear relationship was found between enzyme concentration and reaction rate. The reaction rate was also proportional to the substrate concentration. Inhibition of the reaction was observed at high substrate concentrations. The NAT levels in the liver and kidney of rat, rabbit, mouse and man were measured using this procedure. The tissues of dog failed to acetylate this substrate. The method is applicable to kinetic studies such as the analysis of inhibition reactions with o-phenanthroline and p-chloromercuribenzoate.