Simultaneous determination of nabumetone and its principal metabolite in medicines and human urine by time-resolved fluorescence.

A simple fluorescent methodology for the simultaneous determination of nabumetone and its main metabolite, 6-methoxy-2-naphthylacetic acid (6-MNA), in pharmaceutical preparations and human urine is proposed. Due to the strong overlapping between the fluorescence spectra of both analytes, the use of fluorescence decay curves to resolve their mixture is proposed, since these curves are more selective. Values of dependent instrumental variables affecting the signal-to-noise ratio were fixed using a simplex optimization procedure. A factorial design with three levels per factor coupled to a central composite design was selected to obtain a calibration matrix of thirteen standards plus one blank sample that was processed using a partial least-squares (PLS) analysis. In order to assess the goodness of the proposed method, a prediction set of ten synthetic samples was analyzed, obtaining recovery percentages between 97 and 105%. Limits of detection, calculated by means of a new criterion, were 0.96 µg L(-1) and 0.88 µg L(-1) for nabumetone and 6-MNA, respectively. The method was also tested in the pharmaceutical preparation Relif, which contains nabumetone, obtaining recovery percentages close to 100%. Finally, the simultaneous determination of both analytes in human urine samples was successfully carried out by the PLS-analysis of a matrix of fifteen standards plus four analyte blanks and the use of the standard addition technique. Although urine shows native fluorescence, no extraction method or prior separation of the analytes was needed.

Simplex optimization of the variables affecting the micelle-stabilized room temperature phosphorescence of 6-methoxy-2-naphthylacetic acid and its kinetic determination in human urine.

This article reports the kinetic determination of 6-methoxy-2-naphthylacetic acid (6-MNA), the major metabolite of nabumetone, from micelle-stabilized room temperature phosphorescence (MS-RTP) measurements made by using the stopped-flow mixing technique. This methodology allows one to determine analytes in complex matrices without the need for a tedious separation process. It also shortens analysis times substantially. The proposed method uses simplex methodology to optimize the chemical and instrumental variables affecting the phosphorescence. It was applied to the determination of 6-MNA in human urine. The maximum phosphorescence signal is obtained within only 10 s after the sample is prepared. The maximum slope of the kinetic curve, which corresponds to the maximum rate of the phosphorescence development, is measured at lambda(ex)=273 nm and lambda(em)=516 nm. Least-squares regression was used to fit experimental data, and the detection limit, repeatability, and standard deviation for replicate samples were determined.

Simultaneous analysis of naproxen, nabumetone and its major metabolite 6-methoxy-2-naphthylacetic acid in pharmaceuticals and human urine by high-performance liquid chromatography.

A high-performance liquid chromatographic (HPLC) method for simultaneous determination of naproxen (NAP), nabumetone (NAB) and its major metabolite, 6-methoxy-2-naphthylacetic acid (6-MNA), was developed for the application to pharmaceuticals and human urine. Isocratic reversed-phase HPLC was employed for quantitative analysis using triethylamine and 1-heptanesulfonic acid sodium salt (HSA) as ion-pair reagents. Urine samples were purified by solid-phase extraction using Bond-Elut Certify II cartridges containing reversed-phase and anion exchange functionalities. The HPLC assay was carried out using a Wakosil ODS 5C18 column (5 microm, 150 x 4.6 mm, i.d.). The mobile phase consisted of 0.5 g of HSA dissolved in 1,000 ml of a mixture of acetonitrile, water and triethylamine (500:500:1, v/v) adjusted with phosphoric acid to pH 3. The calibration curves of NAP and NAB showed good linearity in the concentration range 32-160 microg/ml with UV detection (270 nm) for pharmaceuticals. In the low concentration ranges (8-96 ng of NAP per ml, 24-288 ng of NAB per ml and 5.6-67.2 ng of 6-MNA per ml), the calibration curves were also obtained with fluorimetric detection (excitation 280 nm, emission 350 nm) for biological fluids. The correlation coefficients were better than 0.999 in all cases. The lower limits of detection (defined as a signal-to-noise ratio of about 3) were approximately 0.3 ng for NAP, 1.5 ng for NAB and 0.2 ng for 6-MNA. The procedure described here is rapid, simple, selective, and is suitable for routine analysis of pharmaceuticals and pharmacokinetic studies in human urine samples.

Disposition and excretion of 6-methoxy-2-naphthylacetic acid, the active metabolite of nabumetone in horses.

OBJECTIVE: To examine, in horses, the disposition and excretion of the active metabolite 6-methoxy-2-naphthylacetic acid (6MNA) of the nonsteroidal anti-inflammatory prodrug nabumetone. DESIGN: Pharmacokinetic analysis of 6MNA after oral administration of nabumetone and IV administration of 6MNA. PROCEDURE: Using a crossover design, 5 horses were orally administered 3.7 mg of nabumetone/kg of body weight. After a 3-week washout period, 4 horses were administered 2.5 mg of 6MNA/kg, IV. RESULTS: Absorption of nabumetone from the gastrointestinal tract and its metabolism to 6MNA had a median appearance half-life of 0.88 hour. The elimination half-life was 11 hours. Area under the plasma concentration time curve for 6MNA after oral administration of nabumetone was 120.6 mg/h/L. A dose of 2.5 mg/kg of 6MNA administered IV resulted in plasma concentration nearly equivalent to that induced by the orally administered dose. Disposition of 6MNA was modeled as a one-compartment, first-order elimination. The area under the plasma concentration time curve for IV administration of 6MNA was 117.0 mg/h/L, and the specific volume of distribution was 0.247 L/kg. The distribution half-life and the elimination half-life were 0.56 and 7.90 hours, respectively. Percentage of total dose recovered in urine for the 36-hour collection period after the oral and IV administrations was 7.4 and 5.3%, respectively. CONCLUSIONS: Metabolism of nabumetone to 6MNA, as reported in other species, also occurs in horses. There were a number of additional metabolites of nabumetone in urine that could not be fully identified and characterized.

Metabolism of isobutylnaphthyl acetic acid in rats: determination of the chemical structures of metabolites.

After oral and intravenous administration of radiolabelled isobutylnaphthyl acetic acid (INAA) to rats two metabolites were isolated from urine and plasma by HPLC. Field desorption, high resolution electron impact mass spectrometry as well as GC-MS after derivatization were used for structure elucidation and identification of the metabolites. The main biotransformation product in rat urine was found to be 5-(2'-hydroxy-2'-methyl-propyl)-1-naphthyl acetic acid (M1). The main metabolite in plasma was derived and was found to be 5-(2'-carboxypropyl)-1-naphthyl acetic acid (M2).

Studies on the metabolism of arylacetic acids. 6. Comparative metabolic conjugation of 1- and 2-naphthylacetic acids in the guinea pig, mouse, hamster and gerbil.

The patterns of metabolic conjugation of the isomeric 1- and 2-naphthylacetic acids have been compared in guinea pig, mouse, hamster and gerbil. Equimolar doses of [carboxy-14C]1- and 2-naphthylacetic acids were given to these species by i.p. injection, their urine collected and urinary metabolites examined by t.l.c. before and after treatment with beta-glucuronidase or mild alkali. 2. Urinary excretion of 14C following administration of 14C-1-naphthylacetic acid was 76-93% of dose in 72 h, the bulk being eliminated in 24 h. Urinary metabolites comprised 1-naphthylacetic-glycine and -glucuronide together with the unchanged acid. 3. Following administration of 14C-2-naphthylacetic acid, some 68-94% of the 14C dose was recovered in the urine in 72 h, with the majority in the 0-24 h urine. All four species excreted 2-naphthylacetyl-glucuronide and -glycine: additionally, 2-naphthylacetyl-taurine was excreted by mouse, gerbil and hamster and the glutamine conjugate was also present in hamster urine.

Studies on the metabolism of arylacetic acids. 7. The influence of varying dose size upon the conjugation pattern of 2-naphthylacetic acid in the guinea pig, mouse and hamster.

1. The influence of dose size upon the metabolic conjugation of 2-naphthylacetic acid with various amino acids and glucuronic acid has been studied in the guinea pig, mouse and hamster. 2. Guinea pigs conjugated 2-naphthylacetic acid with glycine and glucuronic acid. 3. Mice conjugated 2-naphthylacetic acid with glycine, taurine and glucuronic acid. Taurine conjugation had the highest capacity, and both this and the glycine mechanism were saturated at doses above 100 mg/kg. 4. Hamsters utilized glutamine, glycine, taurine and glucuronic acid for the conjugation of 2-naphthylacetic acid. No conjugation pathway was saturated by doses up to 200 mg/kg. 5. The thus-far unique ability of 2-naphthylacetic acid to evoke multiple amino acid conjugations, using the taurine and glutamine mechanisms hitherto unknown in these species, appears to be due to its affinity for previously unrecognized enzyme systems, rather than to saturation of 'normal' pathways revealing novel routes at high doses.

Metabolic conjugation of some carboxylic acids in the horse.

1. 14C-Labelled benzoic acid, salicylic acid and 2-naphthylacetic acid were administered orally to horses, and urinary metabolites investigated by chromatographic and mass spectral techniques. 2. [14C]Benzoic acid (5 mg/kg) was eliminated rapidly in the urine, and quantitatively recovered in 24 h. The major urinary metabolite was hippuric acid (95% of dose) with much smaller amounts of benzoic acid, benzoyl glucuronide and 3-hydroxy-3-phenylpropionic acid. Administration of [ring-D5]benzoic acid together with [14C]benzoic acid to a pony permitted the mass spectral determination of metabolites of the exogenous benzoic acid metabolites in the presence of the same endogenous compounds. 3. [14C]Salicylic acid (35 mg/kg) was eliminated rapidly in the urine, 98% of the 14C dose being excreted in 24 h. The major excretion product was unchanged salicylate (94% of dose). Gentisic acid, salicyluric acid and the ester and ether glucuronides of salicylic acid were very minor metabolites. 4. 2-Naphthyl[14C]acetic acid (2 mg/kg) was excreted very slowly in the urine, with 53 and 77% of the 14C dose being recovered in six days. 2-Naphthylacetylglycine was the major metabolite (26 and 38% dose) and in addition, the glucuronic acid and taurine conjugates were excreted together with unchanged 2-naphthylacetic acid. 5. The study has shown that the horse can utilize glycine, taurine and glucuronic acid for conjugation of xenobiotic carboxylic acids, and that the relative extents of these pathways are governed by the structure of the carboxylic acid.