A novel quality by design approach for development and validation of a green reversed-phase HPLC method with fluorescence detection for the simultaneous determination of lesinurad, febuxostat, and diflunisal: Application to human plasma.

A novel and eco-friendly reversed-phase HPLC method with fluorescence detection was developed for simultaneous estimation of two co-administered antigout drugs (lesinurad and febuxostat) with diflunisal as a nonsteroidal anti-inflammatory drug. Unlike routine methodology, the developed method was optimized using analytical quality by design approach. A full factorial design was applied to optimize the effect of variable factors on chromatographic responses. The chromatographic separation was performed using isocratic elution on the Hypersil BDS C18 column at 40°C. The mobile phase consisted of acetonitrile:potassium phosphate buffer (30.0 mM; pH 5.5, 32.2:67.8% v/v) pumped at a flow rate of 1.0 mL/min and injection volume of 20.0 µL was employed. The proposed method was able to separate the ternary mixture in <10 min. The calibration curves of diflunisal, lesinurad, and febuxostat were linear over concentration ranges of 50.0-500.0, 50.0-700.0, and 20.0-700.0 ng/mL, respectively. Recovery percentages ranging from 98.1 to 101.3% with % relative standard deviation of <2% were obtained upon spiking to human plasma samples, indicating high bioanalytical applicability. Furthermore, the method was found to be excellent green when it was assessed according to Green Analytical Procedure Index and analytical Eco-Scale guidelines.

Sensitive and selective determination of diflunisal in human plasma by LC-MS.

A reliable and sensitive liquid chromatography-tandem mass spectrometry assay has been proposed for the selective determination of diflunisal in the presence of its glucuronide metabolites. The analyte and clofibric acid as internal standard (IS) are extracted from 50 µL of human plasma by solid-phase extraction. Chromatographic separation is conducted on a Prodigy ODS 3V column (150 × 4.6 mm, 5 µm) under isocratic conditions. The possible interference of acyl glucuronide and phenolic glucuronide, the two major inactive metabolites of diflunisal, is also checked in plasma samples. Detection of the analyte and IS is achieved by tandem mass spectrometry, operating in negative ionization and multiple reaction monitoring acquisition mode. The limits of detection and quantitation of the method are 0.10 and 1.00 µg/mL, respectively, with a linear dynamic range of 1.00-160 µg/mL for diflunisal. The intra-batch and inter-batch precision (percent coefficient of variation) is ≤4.2% and the mean recovery is >92% for diflunisal across quality control levels. The method is successfully applied to a bioequivalence study of a 500 mg diflunisal tablet formulation in 30 healthy Indian male subjects under fasting conditions. The reproducibility in the measurement of study data is demonstrated by the reanalysis of 120 incurred samples.

Simultaneous determination of two anti-inflammatory drugs in serum using isopotential fluorimetry.

The simultaneous determination of 6-methoxy-2-naphthylacetic acid (6MNA) and diflunisal in serum samples using the combination of matrix isopotential synchronous fluorescence (MISF) and first derivative technique is proposed. 6MNA and diflunisal exhibit overlapped spectra and serum produces background fluorescence that precludes the direct determination of these anti-inflammatory drugs by conventional fluorimetry. This method provides good analytical results for determination of compounds in samples with unknown background fluorescence. The method was applied for the simultaneous determination of 6MNA and diflunisal in serum samples at concentrations between 20-200 and 100-1000 ng mL(-1), respectively, by means of absolute values of first derivative of synchronous scan at 247.9/364.0 and 262.6/392.4 nm for 6MNA and diflunisal, respectively. In order to obtain maximum sensitivity and adequate selectivity, factors affecting fluorescence intensity were studied. As a result, the analyses were performed in water at a pH of 7.2, adjusted by using sodium dihydrogen phosphate/hydrogen phosphate (0.1M) as a buffer solution. Serum samples were diluted 200 times. Analytical parameters of the proposed method were calculated according to the error propagation theory. The limit of detection calculated according to Clayton was 15.8 and 63.0 ng mL(-1) for 6MNA and diflunisal, respectively. The sensitivity, repeatability and reproducibility achieved with the proposed method were adequate for the determination of these anti-inflammatory agents in serum samples.

Direct determination of closely overlapping drug mixtures of diflunisal and salicylic acid in serum by means of derivative matrix isopotential synchronous fluorescence spectrometry.

A direct method for the simultaneous fluorimetric determination of two anti-inflammatory drugs in serum is proposed. The combination of matrix isopotential synchronous fluorescence (MISF) and first derivative technique provides good analytical results and permits the simultaneous determination of diflunisal and salicylic acid in human serum. MISF spectra are obtained by calculating the isopotential trajectory in the three-dimensional fluorescence spectrum for a serum solution. In the spectral contour, the trajectory is taken to be the portion of the line that passes by the fluorescence maxima of both compounds ensuring a sensitivity level similar to that of a direct determination in absence of background fluorescence. Analysis was carried out in water using a pH of 7.2 provides by 0.1 M sodium dihydrogen phosphate buffer. Serum samples are diluted 100 times and provide linear calibration plots at diflunisal and salicylic acid concentrations up to 800 ng mL(-1). The goodness of the analytical signal was checked by using variance analysis. Signals recorded throughout the calibration range were subjected to three calibrations per each analyte, both in the absence and in the presence of variable amounts of the other analyte. Differences between individual calibrations and slopes were compared with those within individual calibrations. Based on the results, diflunisal and salicylic acid can be accurately quantified in the presence of each other. The limit of detection calculated according to Clayton who uses error propagation throughout the calibration curve and a non-centralized security factor was 36.8 and 37.3 ng mL(-1) for diflunisal and salicylic acid, respectively.

Selective binding to transthyretin and tetramer stabilization in serum from patients with familial amyloidotic polyneuropathy by an iodinated diflunisal derivative.

In familial amyloidotic polyneuropathy, TTR (transthyretin) variants are deposited as amyloid fibrils. It is thought that this process involves TTR tetramer dissociation, which leads to partially unfolded monomers that aggregate and polymerize into amyloid fibrils. This process can be counteracted by stabilization of the tetramer. Several small compounds, such as diclofenac, diflunisal and flufenamic acid, have been reported to bind to TTR in vitro, in the T4 (thyroxine) binding channel that runs through the TTR tetramer, and consequently are considered to stabilize TTR. However, if these agents bind plasma proteins other than TTR, decreased drug availability will occur, compromising their use as therapeutic agents for TTR amyloidosis. In the present work, we compared the action of these compounds and of new derivatives designed to increase both selectivity of binding to TTR and inhibitory potency in relation to TTR amyloid fibril formation. We found two diflunisal derivatives that, in contrast with diclofenac, flufenamic acid and diflunisal, displaced T4 from TTR in plasma preferentially over binding to albumin and thyroxine binding globulin. The same diflunisal derivatives also had a stabilizing effect on TTR tetramers in plasma, as studied by isoelectric focusing of whole plasma under semi-denaturing conditions. In addition, by transmission electron microscopy, we demonstrated that, in contrast with other proposed TTR stabilizers (namely diclofenac, flufenamic acid and diflunisal), one of the diflunisal derivatives tested efficiently inhibited TTR aggregation. Taken together, our ex vivo and in vitro studies present evidence for the selectivity and efficiency of novel diflunisal derivates as TTR stabilizers and as inhibitors of fibril formation.

Evaluation of several solid phase extraction liquid chromatography/tandem mass spectrometry on-line configurations for high-throughput analysis of acidic and basic drugs in rat plasma.

Several configurations using 6- and 10-port switching valves were studied for high flow, on-line extraction of rat plasma coupled to an electrospray triple quadrupole mass spectrometer. Each plasma sample was diluted 1:1 with an aqueous internal standard solution. The sample was injected into a 2.1 x 20 mm cartridge column packed with 25 microm divinylbenzene/N-vinylpyrrolidone packing using 100% aqueous mobile phase at 4 mL/min. After sample loading and sample cleanup, the analytes were eluted from the extraction column with a 1.0-min gradient at 0.4 mL/min. The samples were either analyzed directly after elution from the extraction column or after additional separation using a short high performance liquid chromatography (HPLC) column. The different configurations were tested using an acidic drug (diflunisal) and a basic drug (clemastine) in rat plasma. On-line analysis was performed by injecting 200 microL of diluted plasma. The mass spectrometer was operated in the multiple reaction monitoring (MRM) mode. All calibration standards gave relative standard deviations (RSDs) below 5%. The total time per sample was 3 min.

[Determination of diflunisal in plasma by RP-HPLC after solid-liquid extraction].

A RP-HPLC method was developed with solid-liquid extraction technique. Plasma sample was extracted on a macroreticular resin cartridge with methanol--glacial acetic acid (99:1) as elution solvent. After extraction, the assay was carried out on a Spherisorb C18 column with p-phenylphenol as internal standard. The mobile phase is a mixture of methanol--water--glacial acetic acid (66:30:4). UV detection was performed at 250 nm. The flow rate was 1.0 ml.min-1. A good linearity was found at the concentration range from 0.5 to 100 micrograms.ml-1, with the lowest detection limit 0.02 microgram.ml-1 (S/N = 2). The extraction and method recoveries were 91.65% and 97.25% respectively, while the RSD for the within-day and between-day precision were all less than 10%. The above method was applied to determine the plasma concentration of difunisal in three human volunteers after a single oral dosage of 300 mg. Two hours after administration, the plasma concentration of diflunisal reached maximum level. A two compartment method was used to study the pharmacokinetic parameters. The T1/2 alpha and T1/2 beta were 1.40 h and 17.85 h, respectively.

Conjugation-deconjugation cycling of diflunisal via beta-glucuronidase catalyzed hydrolysis of its acyl glucuronide in the rat.

The role of beta-glucuronidase catalyzed hydrolysis of glucuronides on the in vivo disposition kinetics of xenobiotics was studied in the rat. The metabolic disposition kinetics of diflunisal, a compound undergoing transformation to an acyl and phenyl glucuronide, were studied in rats under control conditions and following administration of D-glucaro-1,4-lactone, a potent and specific beta-glucuronidase inhibitor. D-glucaro-1,4-lactone treatment resulted in a significant decrease in beta-glucuronidase activity in plasma, urine, and hepatic microsomes. Total (i.e. urinary and biliary) recovery of the acyl glucuronide following i.v. injection of diflunisal (10 mg/kg) was significantly higher in D-glucaro-1,4-lactone treated rats (41 +/- 3%, n=6) compared to control rats (29 +/- 2%, n=6) whereas for diflunisal phenyl glucuronide this total recovery was very similar in both groups of rats (16.0 +/- 1.0% vs. 18.0 +/- 0.2%, n=6, respectively). The partial clearance of diflunisal associated with the formation of the acyl glucuronide was significantly higher in D-glucaro-1,4-lactone treated rats (0.413 +/- 0.024 ml/min/kg) compared to control animals (0.269 +/- 0.042 ml/min/kg). The partial clearance related to the formation of the phenyl glucuronide, on the contrary, was not significantly affected by D-glucaro-1,4-lactone treatment. These results shows that the in vivo glucuronidation of diflunisal to the acyl glucuronide, unlike diflunisal glucuronidation to the phenyl glucuronide, is subject to a futile conjugation-deconjugation cycle. Such futile cycling may have significant therapeutic and toxic implications.

Vesico-hepato-renal cycling of acidic drugs via their reactive acyl glucuronide metabolites? Studies with diflunisal in rats.

1. Deconjugation-reconjugation cycling of acidic drugs is known to occur in vivo via the hydrolysis of their reactive acyl glucuronide metabolites during their circulation in the blood (systemic cycling) or during their passage through the gut after biliary excretion (enterohepatic cycling). Whether such cycling occurs after renal excretion via hydrolysis in the urinary bladder followed by absorption of liberated drug (vesico-hepato-renal cycling) was investigated in rats using diflunisal (DF) and its acyl glucuronide (DFAG) as model compounds. 2. After administration of DF (1 mg/0.5 mL buffer, pH 7) into the bladder of anaesthetized bile-exteriorized rats, DF appeared rapidly in plasma, achieving peak concentrations of 7 micrograms/mL at 1 h. At 4 h, 30% of the dose was recovered as metabolites, mainly DFAG and DF phenolic glucuronide (DFPG) in bile, while 30% was recovered as unchanged DF from the bladder. 3. By contrast, after intravesical administration of an equimolar amount of DFAG at pH 7 or 5, DFAG itself was not detectable in plasma. Plasma concentrations of DF were barely detectable, with only approximately 1% of the administered dose recovered as metabolites in bile. 4. The data thus show that, although DF itself undergoes facile absorption from the urinary bladder of healthy rats, vesico-hepato-renal cycling of DF via DFAG appears to be of only minor quantitative importance.

Effect of cyclodextrins on protein binding of drugs: the diflunisal/hydroxypropyl-beta-cyclodextrin model case.

The binding of diflunisal to hydroxypropyl-beta-cyclodextrin (HP beta CD), bovine serum albumin (BSA), human serum albumin (HSA), normal human plasma, and mixed solutions of HP beta CD/protein was studied at 25 degrees C, pH 7.4, by potentiometry using an electrode selective to diflunisal. The experimental data for diflunisal/HP beta CD fit well to the 1:1 binding model. The binding of diflunisal with each of the studied proteins was compatible with a model having two independent classes of binding sites. The binding of diflunisal in mixed solutions HP beta CD/BSA, HP beta CD/HSA, and HP beta CD/plasma increased considerably when the HP beta CD concentration was increased. The binding behavior of the two biomolecules in the mixed solutions of HP beta CD/BSA or HP beta CD/HSA was described with an "additive" model formulated on the basis of the estimates of the binding parameters of diflunisal derived from the separate experiments with each one of the binders tested. The lower than theoretical binding observed in HP beta CD/plasma solutions was ascribed to the competitive displacement of diflunisal from the HP beta CD cavity by plasma cholesterol.

Studies on the renal excretion of the acyl glucuronide, phenolic glucuronide and sulphate conjugates of diflunisal.

1. In five healthy male volunteers given multiple doses of diflunisal (DF), renal clearances (CLR) of the acyl glucuronide (DAG), phenolic glucuronide (DPG) and sulphate (DS) conjugates were about 42, 25 and 13 ml min-1, respectively. 2. These relatively low CLR values are probably due largely to the very high plasma protein binding of the conjugates, found in vitro to be 99.0%, 97.8% and 99.45%, respectively. 3. Thus glomerular filtration plays the minor and active tubular secretion the major role in renal excretion of the three conjugates. 4. This conclusion was supported by the effect of probenecid co-administration, which decreased CLR of DAG and DPG by about 70%. CLR for DS could not be calculated when probenecid was co-administered (because of interference by probenecid metabolites in the analysis of DS in urine). 5. Water-induced diuresis had no effect on CLR of the DF conjugates, consistent with tubular reabsorption being negligible.

Studies on the reactivity of acyl glucuronides--IV. Covalent binding of diflunisal to tissues of the rat.

Acyl glucuronides have been shown to be reactive electrophilic metabolites capable of undergoing hydrolysis, rearrangement (isomerization via acyl migration) and covalent binding reactions to plasma protein. The present study was undertaken to explore the occurrence and extent of in vivo formation of covalent adducts of diflunisal (DF), a salicylate derivative which forms a reactive acyl glucuronide, with tissues and plasma protein of rats. Groups of rats were given 50 mg DF/kg i.v. twice daily for periods of up to 7 days. Steady state plasma concentrations of reversibly bound DF and its conjugates (as measured 6 hr after a dose) were achieved by the third day of dosing. T 1/2 values after cessation of dosing were about 5-10 hr. By contrast, covalent DF-tissue adducts steadily accumulated over the 7-day dosing period. Maximum concentrations, measured 6 hr after the last dose, were 4.8 (liver), 1.0 (kidney), 0.74 (plasma), 0.26 (small intestine minus contents), 0.27 (large intestine minus contents) and 0.20 (skeletal muscle) microgram DF/g tissue or/mL plasma. T 1/2 values of about 50, 67, 18, 38 and 43 hr were obtained for liver, kidney, plasma and small and large intestine (respectively) after cessation of dosing. Thus, the study of acyl glucuronide reactivity and the question of any derived toxicity or immune responses should consider the formation of long-lived adducts in tissues as well as in plasma.

Interactions between oleic acid and drug competitors influence specific binding of thyroxine in serum.

Long chain nonesterified fatty acids and various drugs may share albumin-binding sites in common. We questioned whether serum binding of T4 could be indirectly influenced by displacement of drug competitors from these sites by nonesterified fatty acids. The influence of oleic acid on drug-induced inhibition of [125I]T4 binding was measured by equilibrium dialysis, using undiluted serum in order to avoid dilution-related artefacts. Oleic acid (1 mmol/L) alone did not inhibit serum protein binding of T4, but this concentration augmented the inhibitory effects on T4 binding of diflunisal, mefenamic acid, meclofenamic acid, and aspirin. This effect increased with increasing concentrations of mefenamic acid, meclofenamic acid, and furosemide. The T4-displacing effect of fenclofenac was not augmented by oleic acid. The mechanism of these interactions was studied by examining 1) oleic acid effects on drug binding, and 2) drug effects on oleic acid binding in undiluted serum. Increments in added oleic acid (0.5-2.0 mmol/L) progressively increased the mean unbound fractions of [14C]aspirin, [14C] diflunisal, and [14C]furosemide, but did not displace [14C]fenclofenac. At the relevant total and free drug concentrations, the inhibitory effect of oleic acid on drug binding and its influence on drug-induced displacement of T4 were concordant in the order: meclofenamic acid greater than aspirin greater than mefenamic acid greater than diflunisal greater than furosemide greater than fenclofenac. In contrast, drug-induced increases in the unbound fraction of [14C]oleic acid did not correlate with augmentation of T4 displacement. We conclude that synergistic effects of oleic acid and drugs on T4 binding result from drug displacement by oleic acid, rather than the reverse effect. Hence, substances that increase the unbound concentration of a competitor by displacing it from albumin can increase its T4-displacing potency. Interactions between various ligands may exert a greater hormone-displacing effect than the sum of each alone.

Pharmacokinetics of diflunisal in patients.

There are few pharmacokinetic data available on the disposition of diflunisal in patients; this study, therefore, looked at the oral absorption, distribution and elimination of this drug. Ten pharmacokinetic profiles obtained from 8 patients showed a maximum plasma diflunisal concentration of 62.0 +/- 26.5 mg/L after 2.76 +/- 1.87 hours, and an area under the plasma concentration-time curve (AUC) of 678.3 +/- 362.3 mg/L.h. Significant intra- and intersubject variability was observed in this group of patients. Analysis of biliary secretion of diflunisal in 4 patients suggested a biliary elimination and subsequent enterohepatic circulation ranging between 2.4 and 15.1%. The AUC for diflunisal in synovial fluid collected from 66 patients was about 70% of that for plasma. In none of 28 patients studied could any trace of diflunisal be observed in cerebrospinal fluid, even though the sensitivity of the assay allowed detection of concentrations as low as 0.01 mg/L.

Liquid chromatography with amperometric detection for the determination of non-steroidal anti-inflammatory drugs in plasma.

A simple and sensitive liquid chromatographic method with electrochemical detection is described for the quantitative determination of the non-steroidal anti-inflammatory drugs diflunisal, indomethacin, naproxen, piroxicam and sulindac in human plasma. Isolation of the drug from the biological fluid is achieved using a Sep-pak RP18 cartridge. Separation of plasma components occurs on a reversed-phase C18 column with a mobile phase consisting of methanol-water-phosphate buffer. For the amperometric detection the potential of +0.9 V was set on the working electrode. The detection limit of the assay is 10-20 ng/ml. The method showed good concordance for plasma samples containing the drugs (r = 0.999) and can be readily utilized for clinical pharmacokinetic studies.

Effects of blockage of urine and/or bile flow on diflunisal conjugation and disposition in rats.

1. The effects of surgical blockage of either or both of the urinary and biliary excretion routes on the elimination of diflunisal (DF) and its conjugates were investigated in pentobarbitone-anaesthetized rats given DF at 10 mg/kg i.v. 2. In control animals the acyl glucuronide and phenolic glucuronide conjugates were excreted predominantly in bile, whereas the sulphate conjugate was eliminated almost exclusively in urine. 3. Bilateral ureter ligation had little effect on DF elimination, except for accumulation of the sulphate conjugate in plasma. Compensatory biliary excretion did not occur. 4. Total plasma clearance of DF decreased from 1.01 to 0.68 ml/min per kg following bile duct ligation. Plasma concentrations and urinary excretion of the glucuronides were elevated. 5. In rats with blockage of both urinary and biliary excretion routes, total plasma clearance of DF decreased to 0.59 ml/min per kg. Both the sulphate and phenolic glucuronide conjugates accumulated in plasma, whereas the acyl glucuronide peaked at 30 min and then declined in parallel with DF. The latter result indicates systemic instability of DF acyl glucuronide with hydrolytic regeneration of DF as the likely major consequence.

The effect of diethyl ether, pentobarbitone and urethane anaesthesia on diflunisal conjugation and disposition in rats.

1. The disposition of diflunisal (DF) at 10 mg/kg i.v. was investigated over 4 h in bile-exteriorized male rats continuously anaesthetized with (a) diethyl ether inhalation (as required), (b) pentobarbitone sodium i.p. (55 mg/kg initially), (c) urethane i.p. (1500 mg/kg initially) or (d) urethane i.v. (750 mg/kg initially), and compared to that obtained in conscious rats. 2. Diethyl ether decreased the plasma clearance of DF to about 30% of control values, by inhibition of both glucuronidation and sulphation of DF. 3. Pentobarbitone anaesthesia caused only modest inhibition of DF elimination, with plasma clearance decreased to about 80% of control values. 4. Plasma profiles and biliary recovery of DF and its conjugates were little altered by urethane i.p. anaesthesia, but urinary recovery was low and variable because of the nearanuria produced by urethane via this administration route. 5. Urinary recovery of DF and its conjugates was satisfactory in rats given urethane i.v., but tissue distribution of DF was substantially decreased. 6. Pentobarbitone was considered to interfere least with DF disposition at the 10 mg/kg dose, and was selected as the most suitable anaesthetic agent for ongoing studies of disposition of DF and its conjugates in anaesthetized rats.

Reactivity of diflunisal acyl glucuronide in human and rat plasma and albumin solutions.

Diflunisal acyl glucuronide (DAG) is a major metabolite of diflunisal (DF) in rats and humans. We have investigated the reactivity of DAG, in purified albumin solutions and plasma from both rat and human sources, along three interrelated pathways: rearrangement via acyl migration to yield positional isomers of DAG, hydrolysis of DAG and/or its isomers to liberate DF, and formation of covalent adducts of DF (via DAG and/or its isomers) with plasma protein. Two initial concentrations of DAG (ca. 50 and 10 micrograms DF equivalents/mL) were used throughout. In all incubations, the order of quantitative importance of the reactions was: rearrangement greater than hydrolysis greater than covalent binding. At pH 7.4 and 37 degrees, degradation of DAG in albumin solutions (e.g. half-life ca. 95 min in fatty acid-free human serum albumin) was retarded in comparison to that found in buffer alone (half-life ca. 35 min). Degradation in unbuffered rat and human plasma containing heparin was comparable to that found in buffer. Maximal covalent binding to protein was achieved after 4-8 hr incubation, and was greatest for fatty acid-free human serum albumin (165 ng DF/mg albumin). Thereafter, slow degradation of the adducts was observed. Formation of DF-plasma protein adducts in vivo was also found in rats and humans dosed with DF.

Covalent binding of diflunisal and probenecid to plasma protein in humans: persistence of the adducts in the circulation.

Acyl glucuronide conjugates of carboxylic drugs have been shown over the past decade to be potentially reactive metabolites, undergoing hydrolysis, intramolecular rearrangement and intermolecular transacylation reactions. The present report describes the covalent binding of diflunisal (D) and probenecid (P) to plasma protein of five healthy volunteers who took a 6-day course of oral D with oral P co-administered during the last 2 days. Maximum concentrations of the D- and P-adducts were achieved within one day of cessation of dosing, and were 35 +/- SE 1 and 17 +/- SE 1 ng/mg protein respectively. The D-protein adduct was eliminated from plasma in a biphasic manner, with a terminal half-life of 10.0 +/- SE 0.9 days. In contrast, elimination of the P-protein adduct was monophasic with a half-life of 13.5 +/- SE 0.3 days. The adducts were still measurable in plasma at least one month after the parent reversibly-bound drugs were undetectable. It is not known whether such covalent binding of the drugs, presumably via their acyl glucuronides, could have any biological consequences, such as induction of hypersensitivity reactions.