Development of an eco-friendly fluorescent probe for mefenamic acid sensing in pharmaceuticals and biofluids.

Mefenamic acid, renowned for its analgesic properties, stands as a reliable choice for alleviating mild to moderate pain. However, its versatility extends beyond pain relief, with ongoing research unveiling its promising therapeutic potential across diverse domains. A straightforward, environmentally friendly, and sensitive spectrofluorometric technique has been developed for the precise quantification of the analgesic medication, mefenamic acid. This method relies on the immediate reduction of fluorescence emitted by a probe upon interaction with varying concentrations of the drug. The fluorescent probe utilized, N-phenyl-1-naphthylamine (NPNA), was synthesized in a single step, and the fluorescence intensities were measured at 480 nm using synchronous fluorescence spectroscopy with a wavelength difference of 200 nm. Temperature variations and lifetime studies indicated that the quenching process was static. The calibration curve exhibited linearity within the concentration range of 0.50-9.00 µg/mL, with a detection limit of 60.00 ng/mL. Various experimental parameters affecting the quenching process were meticulously examined and optimized. The proposed technique was successfully applied to determine mefenamic acid in pharmaceutical formulations, plasma, and urine, yielding excellent recoveries ranging from 98% to 100.5%. The greenness of the developed method was evaluated using three metrics: the Analytical Eco-scale, AGREE, and the Green Analytical Procedure Index.

In-situ synthesis of nanocubic cobalt oxide @ graphene oxide nanocomposite reinforced hollow fiber-solid phase microextraction for enrichment of non-steroidal anti-inflammatory drugs from human urine prior to their quantification via high-performance liquid chromatography-ultraviolet detection.

The in-situ synthesis and application of nanocubic Co3O4-coated graphene oxide (Co3O4@ GO) was introduced for the first time to present a cost-effective, stable and convenient operation and a simple device for hollow fiber solid-phase microextraction (HF-SPME) of four selected nonsteroidal anti-inflammatory drugs (NSAIDs) including diclofenac, mefenamic acid, ibuprofen and indomethacin. The extracted analytes were desorbed by an appropriate organic solvent and analyzed via high-performance liquid chromatography-ultraviolet detection (HPLC-UV). The prepared sorbent was approved using different characterization methods such as X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX). The variables effective on the Co3O4@GO-HF-SPME method including extraction time, desorption time, desorption solvent volume, sample pH, stirring rate and ionic strength were screened via Plackett-Burman design and then optimized by Box-Behnken design. Under optimal condition, the calibration curves were linear within the range of 1.0-200.0 µg L-1 of analyte concentration with detection limits of 0.18-1.1 µg L-1 and the relative standard deviations less than 10.1%. The limits of quantification (LOQs) were in the range of 0.60-3.67 µg L-1. Matrix effect was not observed with this method; therefore, standard addition is not necessary for quantification of target compounds. The enrichment factors were obtained in the range of 49-68. The relative recoveries of the urine sample analysis were calculated in the range of 93-102%. Finally, the presented method exhibited good sensitivity, excellent repeatability, high reusability and acceptable precision, which will be a promising method to analyze various nonsteroidal anti-inflammatory drugs in urine samples.

Nanocrystalline cellulose as a biotemplate for preparation of porous titania thin film as a sorbent for thin film microextraction of ketorolac, meloxicam, diclofenac and mefenamic acid.

The present study included the procedure of preparing porous titania thin film using a direct nanocrystalline cellulose templating (NCC) as a bio-template. The microextraction applicability of the porous film was investigated by thin film microextraction (TFME) of the nonsteroidal anti-inflammatory drugs (NSAIDs) including ketorolac, meloxicam, diclofenac and mefenamic acid from different types of urine sample. The extracted NSAIDs were analyzed by HPLC-UV. Under optimum conditions, the calibration curves were linear within the range of 1.0-500 µg L-1 (2.0-200 µg L-1 for ketorolac, 2.0-500 µg L-1 for meloxicam, 1.0-200 µg L-1 for diclofenac and 1.0-200 µg L-1 for mefenamic acid). The limit of detection was found to be between 0.2 and 0.5 µg L-1. The calculated intra- and inter-day relative standard deviations RSDs% (n = 3) at concentration level of 10 µg L-1 were less than 6.3% and 6.0%, respectively. Finally, the method was successfully applied to determine selected NSAIDs in urine samples from different human individuals.

Application of hydrophobic deep eutectic solvent as the carrier for ferrofluid: A novel strategy for pre-concentration and determination of mefenamic acid in human urine samples by high performance liquid chromatography under experimental design optimization.

In this study, ferrofluid of magnetic nanoparticles, oleic acid (OA), combined with hydrophobic deep eutectic solvent (Fe3O4 [iron oxide]-OA-DES) was prepared and coupled with vortex-assisted liquid phase microextraction, to pre-concentrate and determine mefenamic acid (MFA) in urine samples, following high performance liquid chromatography (HPLC). With regard to Fe3O4 nanoparticles, all sorbents were identified by SEM, TEM, and XRD. In addition, the influence of different parameters such as pH, ferrofluid volume, ionic strong (NaCl concentration %), and vortex time was investigated and optimized by response surface methodology (RSM) and central composite design (CCD). Maximum MFA extraction predicted by RSM (97.66) was achieved at pH 4.0, 60 µL of ferrofluid, 3%w/v NaCl, and 7 min vortex time with desirability of 1.0. Under the optimized conditions, the limits of detection (LOD) and the limit of quantification (LOQ) were 1.351 ng mL-1 and 4.575 ng mL-1, respectively. The intra-day and inter-day precision (RSD [relative standard deviation]%) in the concentrations of 50, 300, and 700 ng mL-1 of MFA were less than 3.6% and 4.2%, respectively. The method developed in our study could successfully determine MFA in urine samples. The MFA microextraction in urine samples were between 80.25 and 97.44% with RSD% less than 4.60%. Inclusion of ferrofluid in a miniaturized method for sample preparation is proportional to the achievement of high microextraction percentage using green analytical method.

Dual functional monomers modified magnetic adsorbent for the enrichment of non-steroidal anti-inflammatory drugs in water and urine samples.

According to the molecular properties of non-steroidal anti-inflammatory drugs (NSADs), a new adsorbent for magnetic solid phase extraction (MSPE) was designed and synthesized. Triethyl-(4-vinylbenzyl)aminium chloride and 4-vinylbenzeneboronic acid were utilized as dual functional monomers to copolymerize with divinylbenzene on the surface of pre-modified Fe3O4 nanoparticles. The prepared magnetic adsorbent (Fe3O4@TCVA) was characterized by elemental analysis, Fourier transform infrared, scanning electron microscopy, transmission electron microscopy and vibrating sample magnetometer. Due to the abundant boronic acid, quaternary amine and phenyl groups, the Fe3O4@TCVA displayed satisfactory extraction performance for target NSADs (diclofenac acid, ibuprofen and mefenamic acid) by means of B-N coordination, anion-exchange, π-π and hydrophobic interactions. Under the optimized conditions, the Fe3O4@TCVA/MSPE was combined with high-performance liquid chromatography with diode array detection (HPLC-DAD) to sensitively analyze NSADs in water and human urine samples. Results indicated that the limits of detection for water and urine samples were in the ranges of 0.014-0.031 µg/L and 0.029-0.11 µg/L, respectively. The relative standard deviations for the intra-day and inter-day assay variability were below 10%. The applicability of the proposed Fe3O4@TCVA/MSPE-HPLC-DAD method was demonstrated by the successful extraction and quantification of trace levels of NSADs in real water and human urine samples. Satisfactory spiked recovery and reproducibility were achieved.

Sensors based on ruthenium-doped TiO2 nanoparticles loaded into multi-walled carbon nanotubes for the detection of flufenamic acid and mefenamic acid.

Recent advances to utilize two or more nanoparticles for developing novel sensors with superior sensitivity have spurred advanced detection limits even at low concentrations. In this research, a blend of rutheniumdoped TiO2 (Ru-TiO2) nanoparticles and multiwalled carbon nanotubes (MWCNTs) loaded into carbon paste matrix to fabricate a novel Ru-TiO2/MWCNTs-CPE sensor was used for the detection and quantification of flufenamic acid (FFA) and mefenamic acid (MFA) drugs. The surface morphology of Ru-TiO2 was assessed by scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), transmission electron microscopy (TEM), X-ray diffraction (XRD) and atomic force microscopy (AFM). Sensitivity and selectivity of the electrode was improved at the Ru-TiO2/MWCNTs modified CPE compared to nascent CPE due to the amazing surface distinctive characteristics of the modifier at pH 5.0. The effect of concentration of the modifier, pH, pre-concentration time, sweep rate and concentration on signal enhancement of FFA and MFA was studied. The square wave voltammetry (SWV) currents are linearly related in the concentration range of 0.01 µM-0.9 µM with the detection limit values of 0.68 nM for FFA and 0.45 nM for MFA, respectively. The developed electrode assembly was used for the quantification of both the drug analytes in human urine samples.

Isoform-specific therapeutic control of sulfonation in humans.

The activities of hundreds, perhaps thousands, of metabolites are regulated by human cytosolic sulfotransferases (SULTs) - a 13-member family of disease relevant enzymes that catalyze transfer of the sulfuryl moiety (-SO3) from PAPS (3'-phosphoadenosine 5'-phosphosulfonate) to the hydroxyls and amines of acceptors. SULTs harbor two independent allosteric sites, one of which, the focus of this work, binds non-steroidal anti-inflammatory drugs (NSAIDs). The structure of the first NSAID-binding site - that of SULT1A1 - was elucidated recently and homology modeling suggest that variants of the site are present in all SULT isoforms. The objective of the current study was to assess whether the NSAID-binding site can be used to regulate sulfuryl transfer in humans in an isoform specific manner. Mefenamic acid (Mef) is a potent (Ki 27 nM) NSAID-inhibitor of SULT1A1 - the predominant SULT isoform in small intestine and liver. Acetaminophen (APAP), a SULT1A1 specific substrate, is extensively sulfonated in humans. Dehydroepiandrosterone (DHEA) is specific for SULT2A1, which we show here is insensitive to Mef inhibition. APAP and DHEA sulfonates are readily quantified in urine and thus the effects of Mef on APAP and DHEA sulfonation could be studied non-invasively. Compounds were given orally in a single therapeutic dose to a healthy, adult male human with a typical APAP-metabolite profile. Mef profoundly decreased APAP sulfonation during first pass metabolism and substantially decreased systemic APAP sulfonation without influencing DHEA sulfonation; thus, it appears the NSAID site can be used to control sulfonation in humans in a SULT-isoform specific manner.

Development of electrochemically controlled packed-in-tube solid phase microextraction method for sensitive analysis of acidic drugs in biological samples.

In the present research, for the first time, a novel "packed-in-tube" configuration has been applied to electrochemically controlled in-tube solid phase microextraction, followed by high performance liquid chromatography. In order to prepare a mini packed column, small beads of stainless steel were first placed into the stainless steel column. Then, a nanostructured polypyrrole film was prepared on the internal surface of a stainless steel tube and the surface of stainless steel particles through a facile in-situ electrodeposition method. Filling the column with tiny particles of stainless steel effectively reduces the dead volume of the extraction tube and increases the extraction phase volume. The column was used for separation and preconcentration of diclofenac and mefenamic acid as model analytes from biological samples. Several important factors affecting extraction efficiency, such as extraction and desorption times, flow rates of the sample solution and eluent, and extraction and desorption voltages were investigated and optimized. This method showed good linearity for the drugs in the range of 0.3-200.0 µg L-1, 1.1-200.0 µg L-1, and 1.8-200.0 µg L-1 with coefficients of determination better than 0.9986, 0.9973, and 0.9973 in water, urine, and plasma samples, respectively. Intra- and inter-assay precisions (RSD%, n = 3) were in the range of 2.6-4.8% and 2.9-5.1, respectively, at three concentration levels of 10, 25, and 75 µg L-1. In addition, the limits of detection were in the range of 0.02-0.04 µg L-1. The validated method was successfully applied to the analysis of diclofenac and mefenamic acid in some biological samples. Finally, it is concluded that this method can be a general and reliable alternative to the analysis of ionic compounds in biological matrices.

Coupling of two centrifugeless ultrasound-assisted dispersive solid/liquid phase microextractions as a highly selective, clean, and efficient method for determination of ultra-trace amounts of non-steroidal anti-inflammatory drugs in complicated matrices.

In this work, a new, simple, rapid, and environmentally friendly method with a high sample clean-up capability termed as centrifugeless ultrasound-assisted dispersive micro solid-phase extraction coupled with salting-out ultrasound-assisted liquid-liquid microextraction based on solidification of a floating organic droplet followed by high performance liquid chromatography is introduced for the first time. In this method, the three non-steroidal anti-inflammatory drugs diclofenac, ibuprofen, and mefenamic acid are first extracted based on an effective nanoadsorbent named as the layered double hydroxide-carbon nanotube nanohybrid. The first step provides a rapid and convenient way to separate the adsorbent from the sample matrix by a syringe nanofilter without additional centrifugation. In the next step, which is based upon the salting-out effect, after emulsification in the presence of ultrasonic irradiation, the phase separation is simply achieved through the salting-out phenomenon, and the extracting solvent is suspended on top of the sample solution. Under the optimal experimental conditions including the initial pH value of 6.0, 8.0 mg of the nanohybrid, 3 min ultrasonic time, 100 µL elution solvent (first step), secondary pH value of 3.0, 60 µL of 1-undecanol, 60 s ultrasonic time, and flow rate of 3 mL min-1 (second step), good responses were obtained for diclofenac, ibuprofen, and mefenamic acid in the concentration ranges of 0.8-2000, 0.8-2500, and 0.5-2000 ng mL-1, respectively, with low limits of detection ranging from 0.1 to 0.2 ng mL-1. The intra-day and inter-day precisions for the target analytes at the three concentration levels were in the ranges of 6.1-7.8% and 6.3-8.1%, respectively. The proposed method was also successfully applied to the biological and waste water samples, and excellent recoveries were obtained in the range of 92.9-103.1% even when the matrix was complex.

Magnetic solid phase extraction of mefenamic acid from biological samples based on the formation of mixed hemimicelle aggregates on Fe(3)O(4) nanoparticles prior to its HPLC-UV detection.

A novel and sensitive solid phase extraction method based on the adsorption of cetyltrimethylammonium bromide on the surface of Fe3O4 nanoparticles was developed for extraction and preconcentration of ultra-trace amounts of mefenamic acid in biological fluids. The remarkable properties of Fe3O4 nanoparticles including high surface area and strong magnetization were utilized in this SPE procedure so that a high enrichment factor (98) and satisfactory extraction recoveries (92-99%) were obtained using only 50mg of magnetic adsorbent. Furthermore, a fast separation time (about 15min) was achieved for a large sample volume (200mL) avoiding time-consuming column-passing process of conventional SPE. A comprehensive study on the parameters effecting the extraction recovery such of the amount of surfactant, pH value, the amount of Fe3O4 nanoparticles, sample volume, desorption conditions and ionic strength were also presented. Under the optimum conditions, the method was linear in the 0.2-200ngmL(-1) range and good linearity (r(2)>0.9991) was obtained for all calibration curves. The limit of detection was 0.097 and 0.087ngmL(-1) in plasma and urine samples, respectively. The relative standard deviation (RSD %) for 10 and 50ngmL(-1) of the analyte (n=5) were 1.6% and 2.1% in plasma and 1.2% and 1.9% in urine samples, respectively. Finally, the method was successfully applied to the extraction and preconcentration of mefenamic acid in human plasma and urine samples.

Simultaneous determination of mefenamic and tolfenamic acids in real samples by terbium-sensitized luminescence.

A simple luminescent methodology for the simultaneous determination of mefenamic and tolfenamic acids in pharmaceutical preparations and human urine is proposed. Since the native fluorescence of both analytes is not intense, the method takes advantage of the lanthanide-sensitized luminescence, which provides a higher sensitivity. Due to the strong overlapping between the luminescence spectra of both terbium complexes, the use of luminescence decay curves to resolve mixtures of the analytes is proposed, since these curves are more selective. 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 eight blank samples that was processed using a partial least-squares (PLS) analysis. In order to assess the goodness of the proposed method, a prediction set of synthetic samples was analyzed, obtaining recovery percentages between 90 and 104 %. Limits of detection, calculated by means of a new criterion, were 14.85 µg L(-1) and 15.89 µg L(-1) for tolfenamic and mefenamic acids, respectively. The method was tested in a pharmaceutical preparation containing mefenamic acid, obtaining recovery percentages close to 100 %. Finally, the simultaneous determination of both fenamates in human urine samples was successfully carried out by means of a correction of the above-explained model. No extraction method neither prior separation of the analytes were needed.

Second-order advantage applied to simultaneous spectrofluorimetric determination of paracetamol and mefenamic acid in urine samples.

Second-order advantage of excitation-emission fluorescence measurements was applied to the simultaneous determination of paracetamol (PC) and mefenamic acid (MF) in urine samples. Two drugs were quantified by multivariate curve resolution coupled to alternative least squares (MCR-ALS) in micellar media of sodium dodesyl sulfate (SDS). Experimental conditions including pH and SDS concentration were optimized. Under the optimum conditions, pH 2.0 and 0.05 mol L(-1) of SDS, paracetamol and mefenamic acid were determined in concentration range 4.00-20.00 microg mL(-1) and 0.80-5.00 microg mL(-1), respectively, in urine samples.

Second-order calibration of excitation-emission matrix fluorescence spectra for the determination of N-phenylanthranilic acid derivatives.

A spectrofluorimetric method has been developed for the quantitative determination of mefenamic, flufenamic, and meclofenamic acids in urine samples. The method is based on second-order data multivariate calibration (unfolded partial least squares (unfolded-PLS), multi-way PLS (N-PLS), parallel factor analysis (PARAFAC), self-weighted alternating trilinear decomposition (SWATLD), and bilinear least squares (BLLS)). The analytes were extracted from the urine samples in chloroform prior to the determination. The chloroform extraction was optimized for each analyte, studying the agitation time and the extraction pH, and the optimum values were 10 minutes and pH 3.5, respectively. The concentration ranges in chloroform solution of each of the analytes, used to construct the calibration matrix, were selected in the ranges from 0.15 to 0.8 microg mL-1 for flufenamic and meclofenamic acids and from 0.25 to 3.0 microg mL-1 for mefenamic acid. The combination of chloroform extraction and second-order calibration methods, using the excitation-emission matrices (EEMs) of the three analytes as analytical signals, allowed their simultaneous determination in human urine samples, in the range of approximately 80 mg L-1 to 250 mg L-1, with satisfactory results for all the assayed methods. Improved results over unfolded-PLS and N-PLS were found with PARAFAC, SWATLD, and BLLS, methods that exploit the second-order advantage.

Determinations of fluoroquinolones and nonsteroidal anti-inflammatory drugs in urine by extractive spectrophotometry and photoinduced spectrofluorimetry using multivariate calibration.

Multivariate calibration methods are chemometric tools that may be applied to the analysis of spectroscopic data with multichannel detection. Two procedures, based on spectrophotometric and fluorimetric signals, are reported for the simultaneous determination of two fluoroquinolones (ciprofloxacin and ofloxacin) and two nonsteroidal anti-inflammatory drugs (diclofenac and mefenamic acid) using first- and second-order multivariate calibration methods. In the spectrophotometric method, an extractive procedure into chloroform using trioctylmethylammonium chloride-adogen as counter ion was optimized, with the object of extracting the analytes from urine samples and eliminating matrix interferences. After separation, the absorption spectrum of the organic phase was used as the analytical signal in a partial least squares method. A photoinduced spectrofluorimetric (PIF) method using excitation-emission fluorescence matrices, is proposed, to apply three-way chemometric calibration, with the aim of analyzing ofloxacin, ciprofloxacin, and diclofenac in urine samples without the previous extractive sample-cleaning step. For both procedures, recoveries around 100% were found for all the analytes. However, the PIF three-way chemometric method provides the most sensitive and selective procedure as the urine interferences are modulated using the three-way chemometric technique.

Simultaneous analysis of anthranilic acid derivatives in pharmaceuticals and human urine by high-performance liquid chromatography with isocratic elution.

A high-performance liquid chromatographic (HPLC) method for simultaneous determination of mefenamic acid (MFA), flufenamic acid (FFA) and tolfenamic acid (TFA) is presented for application to pharmaceuticals and human urine. Isocratic reversed-phase HPLC was employed for quantitative analysis using tetra-pentylammonium bromide (TPAB) as an ion-pair reagent. Urine samples were purified by solid-phase extraction using a silica-based strong anion-exchanger, Bond-Elut SAX cartridge. The HPLC assay was carried out using a Wakosil ODS 5C18 column (5 microm, 150x4.6 mm I.D.). The mobile phase consisted of 1.9 g of TPAB dissolved in 1:1 of a mixture of acetic acid-sodium acetate buffer solution, pH 5.0, and acetonitrile (11:9, v/v). The calibration curves of MFA, FFA and TFA showed good linearity in the concentration range of 33-167 microg/ml with a wavelength of 280 nm for pharmaceuticals, and in the low concentration range (1.7-30.1 microg/ml) with a wavelength of 230 nm for biological fluids. The correlation coefficients were better than 0.9999 in all cases. The lower limits of detection (defined as a signal-to-noise ratio of about 3) were approximately 2 ng for MFA, 3.5 ng for FFA and 2.5 ng for TFA. The procedure described here is rapid, simple, selective and is suitable for routine analysis of pharmaceuticals and pharmacokinetic studies in human urine samples.

Determination of flufenamic, meclofenamic and mefenamic acids by capillary electrophoresis using beta-cyclodextrin.

The possibility of separating flufenamic, meclofenamic and mefenamic acids by capillary electrophoresis was studied. The best approach involved combining a suitable pH of the carrier electrolyte (pH 12.0) with the host-guest complexation effects of beta-cyclodextrin. A running buffer consisting of 30 mM phosphate buffer (pH 12.0), 2 mM beta-CD and 10% (v/v) acetonitrile was found to provide a very efficient and stable electrophoresis system for the analysis of fenamic acids by capillary zone electrophoresis. Responses were linear from 0.4 to 40 microg/ml for the three drugs with detection limits of about 0.3 ng/ml. Intra- and inter-day precision values of about 1-2% R.S.D. (n = 11) and 3-4% R.S.D. (n = 30), respectively, were obtained. The method is highly robust and no breakdowns of the current or capillary blockings were observed for several weeks. The general applicability of this rapid CZE procedure (migration times less than 12 min) is demonstrated for several practical samples, including serum, urine and pharmaceuticals.

Urinary excretion of mefenamic acid and its metabolites including their esterglucuronides in preterm infants undergoing mefenamic acid therapy.

Urinary excretion of mefenamic acid (MA) and its two oxidative metabolites, M-I (3'-hydroxymethyl derivative) and M-II (3'-carboxyl derivative), and their glucuronides was investigated in preterm infants undergoing MA therapy. MA was given orally at a dose of 2 mg/kg and the dose was repeated every 24 h a maximum of three times. Urine was collected for up to 5 d after the last dose, and MA and the metabolites were determined by a newly developed HPLC. The cumulative amounts of MA and the metabolites excreted in the urine varied from 7 to 46% of the total dose administered, and were less than those reported in adults and children. Significant correlation was observed between the plasma half-life of MA and the cumulative amount of MA and the metabolites excreted in the urine. These results suggest that long plasma half-lives of MA observed in preterm infants are due mainly to low activity of drug metabolizing enzyme(s). In an infant who received the two regimens of MA therapy about 2 weeks apart, the plasma half-life of MA was shortened and the urinary excretion of the MA metabolites including their glucuronides was greatly increased during this period. It is suggested that the activities of both cytochrome P-450(s) and glucuronyltransferase(s) related to MA metabolism rapidly increased during the first month of the infant's life.

Reactivity of mefenamic acid 1-o-acyl glucuronide with proteins in vitro and ex vivo.

Mefenamic acid is a nonsteroidal anti-inflammatory drug commonly used in analgesia. The use of this drug has been implicated in several cases of nephrotoxicity including acute renal failure and tubulointerstitial nephritis. One theory of drug-induced tubulointerstitial nephritis is that the drug or a derivative of the drug becomes irreversibly bound to certain sites in renal tissue and an immune response is directed against the hapten-host conjugate. Previous studies have shown that in humans the nonsteroidal anti-inflammatory drug mefenamic acid is metabolized by both phase I enzymes and the phase II enzyme family UDP-glucuronosyltransferase. Indeed, three glucuronides were identified and isolated from human urine by semipreparative HPLC after oral administration of mefenamic acid. This study focuses on mefenamic acid glucuronide and further characterizes this acyl glucuronide in terms of stability and its ability to bind irreversibly to proteins. Stability studies of mefenamic acid glucuronide in aqueous buffer highlighted the relative stability of this acyl glucuronide at physiological pH. The half-life at 37 degrees C, pH 7.4, was 16.5 +/- 3.1 hr, which is considerably longer than those reported for many acyl glucuronides. The degradation of mefenamic acid glucuronide was accelerated under alkaline conditions, decreasing the half-life to 5 +/- 1.6 hr at pH 8.0. Mefenamic acid glucuronide, although extremely stable in buffer at physiological pH, was found to bind irreversibly to human serum albumin in vitro. Irreversible binding to cellular proteins in culture was also evident with the addition of mefenamic acid to the heterologous Chinese hamster lung fibroblast cell line V79 expressing the human UDP-glucuronosyltransferase isoenzyme UGT1*02. This binding was directly related to glucuronide formation, because irreversible binding was not evident in the untransfected cell line V79.

Structures of mefenamic acid metabolites from human urine.

Three major metabolites of mefenamic acid were isolated from the urine of a normal adult man receiving mefenamic acid orally. The structures of those metabolites were determined as glucuronides of mefenamic acid, its hydroxymethyl derivative, and its carboxylic acid derivative on the basis of spectral data.