Simultaneous determination of febuxostat and montelukast in human plasma using fabric phase sorptive extraction and high performance liquid chromatography-fluorimetric detection.

In the present work, a new sensitive and selective high-performance liquid chromatography-fluorimetric detection (HPLC-FLD) method was developed and validated to quantify febuxostat (FBX) and montelukast (MON) in human plasma. The developed procedure was successfully applied to a study aimed at evaluating the pharmacokinetic profiles of febuxostat and montelukast in human plasma. A sol-gel poly (caprolactone)-block-poly(dimethylsiloxane)-block-poly(caprolactone) (sol-gel PCAP-PDMS-PCAP) extraction sorbent coated fabric phase sorptive extraction membrane was used in the extraction process. The entire chromatographic analysis was performed with isocratic elution of the composition of the mobile phase (acetonitrile:water, 60:40, v:v, 0.032% glacial acetic acid) on the C18 column. The flow rate is varied during the analysis, particularly from 0.5 mL min-1 at the start and linearly increased to 1.5 mL min-1 in 7 min. The detection and quantification of the analytes was carried out by means of a fluorimetric detector at 320 nm and 350 nm as absorption wavelengths and at 380 and 400 nm as emission wavelengths for FBX and MON, respectively. The calibration curves demonstrated linearity in the range 0.3-10 ng mL-1 and 5-100 ng mL-1 for FBX and MON, respectively, while the LOD and LOQ values were 0.1 and 0.3 ng mL-1 for FBX and 1.5 and 5 ng mL-1 for MON. Intraday and interday RSD% values were found lower than 5.79%. As reported, the method was applied to real plasma samples obtained from a volunteer who was co-administered both the drugs. Pharmacokinetic data reveal that the concentration of both the drugs reaches the plateau approximately at the same time, but exhibits an elimination phase at different rates. This study demonstrated the usefulness of the new method and its applicability in therapeutic drug monitoring (TDM).

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.

Development of LC-MS/MS determination method and backpropagation artificial neural networks pharmacokinetic model of febuxostat in healthy subjects.

WHAT IS KNOWN AND OBJECTIVE: Febuxostat is a well-known drug for treating hyperuricemia and gout. The published methods for determination of febuxostat in human plasma might be unsuitable for high-throughput determination and widespread application. We need to develop a highly selective, sensitive and rapid liquid chromatography-tandem mass spectrometry method. METHODS: The chromatographic separation was achieved on a Hypersil Gold-C18 (2.1 mm × 100 mm, 1.9 µm) column with mobile phase A (Water containing 0.1% formic acid) and mobile phase B (acetonitrile containing 0.1% formic acid). Multiple reaction monitoring (MRM) mode was used for quantification using target ions at m/z 315.3 â†’ m/z 271.3 for febuxostat and m/z 324.3 â†’ m/z 280.3 for Febuxostat-d9 (IS). A backpropagation artificial neural network (BPANN) pharmacokinetic model was constructed by the data of bioequivalence study. RESULTS AND DISCUSSION: After the LC-MS/MS method validated, it was successfully applied to the bioequivalence study of 30 human volunteers under fed condition. The predicted concentrations generated by BPANN model had a high correlation coefficient with experimental values. WHAT IS NEW AND CONCLUSION: A sensitive LC-MS/MS method had been developed and validated for determination of febuxostat in healthy subjects under fed condition, and a BPANN model was developed that can be used to predict the plasma concentration of febuxostat.

Simultaneous determination of colchicine and febuxostat in rat plasma: Application in a rat pharmacokinetic study.

A selective, sensitive and rapid LC-MS/MS method has been developed and validated as per US Food and Drug Administration regulatory guidelines for the simultaneous quantitation of colchicine and febuxostat in rat plasma. Colchicine and febuxostat were extracted from the rat plasma using 10% tert-butyl methyl ether in ethyl acetate using colchicine-d6 as an internal standard (IS). The chromatographic separation of colchicine, febuxostat and the IS was achieved using a mobile phase comprising 5 mm ammonium formate and 0.025% formic acid in acetonitrile (20:80, v/v) in isocratic mode on an Eclipse XDB-C18 column. The injection volume and flow rate were 5.0 µl and 0.9 ml/min, respectively. Colchicine and febuxostat were detected by positive electrospray ionization in multiple reaction monitoring mode using transition pairs (Q1 → Q3) of m/z 400.10 → 358.10 and 317.05 → 261.00, respectively. The assay was linear in the ranges of 0.25-254 and 2.60-622 ng/ml for colchicine and febuxostat, respectively. The inter- and intra-day precision values were 0.58-13.0 and 1.03-4.88% for colchicine and febuxostat, respectively. No matrix or carryover effects were observed during the validation. Both analytes were stable on the bench-top, in the autosampler and in storage (freeze-thaw cycles and long-term storage at -80°C). A pharmacokinetic study in rats was performed to show the applicability of the validated method.

Determination of febuxostat in human plasma by high performance liquid chromatography (HPLC) with fluorescence-detection.

Febuxostat prevents gout attacks by lowering serum urate. Aspects of the pharmacokinetic-pharmacodynamic relationship of febuxostat concentrations to urate in gout patients need further elucidation. In order to undertake these studies, the assay methodology for febuxostat has been enhanced and validated to meet FDA standards. An HPLC method with fluorescence-detection has been modified to increase sensitivity, reduce complexity, shorten the sample preparation process and improve the inter-day coefficient of variation of the lowest quality control sample (0.03 µg/L). Protein in plasma samples (200 µL) is now precipitated with acetonitrile (400 µL) containing the internal standard (2-naphthoic acid). The supernatant is analysed at excitation and emission wavelengths of 320 and 380 nm, respectively as in the previous method. A Luna C18 column (Phenomenex, Australia) at 40 °C with mobile phase of glacial acetic acid (0.032%) in acetonitrile:water (60:40, v:v), an injection volume of 10 µL and a flow rate of 1.5 mL/min is employed. Analysis time is 8 min. Calibration curves in drug-free plasma range from 0.005 to 10.00 µg/mL. Data points are fitted using linear regression with a weighting factor of 1/concentration. The inter-day accuracy and imprecision of the quality control samples (0.0075, 0.015, 3.00 and 9.80 µg/mL) is 90-115% and ≤ 14.5%, respectively.

Identification and structural characterization of febuxostat metabolites in rat serum and urine samples using UHPLC-QTOF/MS.

Febuxostat is a novel nonpurine type of highly selective xanthine oxidoreductase inhibitor. A rapid and sensitive ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry method for simultaneous separation and determination of febuxostat and its metabolites in rat serum and urine was developed at various time points after oral administration to the rats. The febuxostat metabolites were predicted by biotransformation software and transformed to a personal compound database to quickly determine the possible metabolites from the MS1 data. The possibility of the MS/MS fragmentation was calculated by the Molecular Structure Correlator software. As a result, five phase I and two phase II metabolites in rat serum, and seven phase I and three phase II metabolites in rat urine were identified, of which four metabolites (M2, M5, M6, M7) have not been reported before. The metabolite toxicities are predicted, and the results are helpful for the design of new xanthine oxidoreductase inhibitors.

Painless gouty tophus in the nasal bridge: A case report and literature review.

RATIONALE: A gouty tophus, arising from the deposition of monosodium urate crystals (MSU), rarely occurs in the nasal bridge. There have been only 7 documented cases of a gouty tophus in the nasal bridge from 1978 to 2018 in English-language literature. PATIENT CONCERNS: A 65-year-old male had a chief complaint of a lump in the nasal bridge that was slowly growing for over 1 year. DIAGNOSIS: MSU crystals were confirmed through ultrasonography (US) and pathological examinations. INTERVENTIONS: A cosmetically less destructive method, ultrasound-guided fine needle aspiration cytology (FNAC) was used to approach the mass lesion of nasal bridge. OUTCOMES: The diagnosis was confirmed as a gouty tophus without performing a nasal subdermal exploration. LESSONS: This case report is the first use of US with FNAC to approach and diagnosed a gouty tophus in the nasal bridge.

A novel voltammetry offline coupled MALDI/TOF MS characterization of electrochemical reaction products and the voltammetric determination of febuxostat in human plasma.

A simple offline coupling voltammetry-MALDI/TOF MS procedure is presented for studying electrochemical reactions. It was utilized for the characterization of the electro-reduction products of febuxostat in methanolic acetate buffer (0.1 M, pH 5). The MS analysis reveals that the carboxylic and nitrile groups are the electro-reducible groups at -0.9338 and -1.5503 V with the conversion to aldehydic and amino groups, respectively. The developed voltammetric method was validated and applied successfully for the drug determination in pharmaceutical tablets and real plasma samples within the linearity ranges 0.03-2 and 0.4-5 µg mL-1, respectively.

A novel HPLC-DAD method for simultaneous determination of febuxostat and diclofenac in biological samples: pharmacokinetic outcomes.

AIM: To develop a simple HPLC-DAD method for simultaneous determination of febuxostat (FEB) and diclofenac (DIC) in biological samples to assess pharmacokinetic outcomes of their coadministration. Methodology & results: Sample preparation was performed by liquid-liquid extraction. Drugs analysis was done on C18 column using methanol-formic acid pH 2.1 (76:24, v/v) as mobile phase and time-programmed UV detection. Lower limits of quantitation for FEB and DIC were 10 and 20 ng/ml, respectively. Baseline pharmacokinetics were similar to published data on either drug alone. Coadministration led to more than twofold increase in FEB Cmax and AUC together with a reduced hepatic uptake in rats. CONCLUSION: DIC interfered with initial distribution and terminal clearance of FEB potentially due to reduced FEB hepatic uptake.

Innovative HPTLC method with fluorescence detection for assessment of febuxostat-montelukast combination and study of their protective effects against gouty arthritis.

The present study was designed to evaluate the potential protective effects of the cysteinyl leukotriene receptor blocker montelukast (MNK) and the xanthine oxidase inhibitor febuxostat (FBX) and their combination on a model of acute gouty arthritis induced by intraarticular injection of monourate sodium crystal (MUC) injection in rats. Additionally, we established an HPTLC method for the quantitative determination of both drugs simultaneously and applied this method to detect this combination in rat plasma. In this study, the treated male Wistar rats were grouped as follows: a negative control group injected with phosphate buffer saline (PBS) and a positive control group injected with MUC in their tibiotarsal joint. Four groups were treated orally with diclofenac (4 mg kg-1), MNK (10 mg kg-1), FBX (5 mg kg-1) and MNK plus FBX before MUC injection in their tibiotarsal joints. MUC injection in joints without pretreatment led to a progressive significant increase in joint diameter and heavy leucocytic infiltration compared to the PBS-injected joints. Treatment with diclofenac or a combination of MNK and FBX significantly decreased both joint diameters and leucocytic infiltration compared to the MUC group. MNK or FBX treatment attenuated leucocytic infiltration and significantly decreased joint diameters compared to the MUC group. Thus, MNK and FBX can prevent the development of MUC-induced acute gouty arthritis in rats. Also, MNK can be an alternative preventive treatment, particularly in elderly patients who cannot tolerate NSAIDs or corticosteroid. Furthermore, a new thin-layer chromatographic method combined with fluorescence detection was developed and validated for the simultaneous estimation of a mixture of FBX and MNK in spiked human plasma. In this method, we employed TLC aluminium plates precoated with silica gel G 60F254 as the stationary phase and chloroform : methanol (9 : 1, v/v) as the mobile phase. The optimized mobile phase selected for development gives compact bands (Rf values are 0.28 and 0.63 for FBX and MNK, respectively). The emission intensities were measured using a K400 optical filter after excitation at 322 nm. The linear regression data for the calibration plots showed excellent linear relationship (r = 0.9990 and 0.9996 for FBX and MNK, respectively), and the linearity range was 10.0-800.0 ng per band for both drugs. According to ICH-guidelines, this method was validated for precision, accuracy, robustness and selectivity. Also, the limits of detection and quantitation were calculated. In addition, stability studies on the studied mixture were performed. Statistical analysis proved that this method is reproducible and selective for the estimation of a mixture of FBX and MNK.

Fluorimetric determination of febuxostat in dosage forms and in real human plasma via Förster resonance energy transfer.

A rapid, simple, selective and precise fluorimetric method was developed and validated for determination of a selective xanthine oxidase inhibitor; febuxostat (FBX) in pharmaceutical formulations and in human plasma. The proposed method is based on quenching effect of FBX on the fluorescence intensity of terbium (Tb3+ ) through fluorescence resonance energy transfer (FRET) from Tb3+ to FBX. The formed complex was measured at λex. 320 nm/λem. 490 nm against a reagent blank. Fluorescence intensity of Tb3+ was diminished when FBX was added. A linear relationship between the fluorescence quenching value of the formed complex ΔF=FTb3+-FFBX-Tb3+ and the concentration of FBX was investigated. The reaction conditions and the fluorescence spectral properties of the complex have been studied. The linearity range of the developed method was 1.0-16.0 µg/ml. The suggested method was applied successfully for the estimation of FBX in bulk powder, dosage forms and spiked plasma samples with excellent recoveries (96.79-98.89%). In addition, the developed method has been successfully applied for determination of FBX in real plasma samples collected from healthy volunteers with good recoveries (82.06-85.65%). All obtained results of the developed method were statistically analyzed and validated according to ICH (International Conference on Harmonization) guidelines.

High-performance thin-layer chromatographic methods for the determination of febuxostat and febuxostat/diclofenac combination in human plasma.

Two simple, sensitive and specific high-performance thin-layer chromatographic (HPTLC) methods were developed for the determination of febuxostat (FEB) individually, and simultaneously with diclofenac (DIC) in human plasma. Method A presents the first HPTLC-ultraviolet attempt for FEB determination in human plasma. FEB was separated from endogenous plasma components (at hRF = 70) with ethyl acetate-methanol-water (9:2:1, v/v) mixture as mobile phase and quantified by densitometry at its λmax (315 nm). Method B is considered the first attempt for the simultaneous determination of FEB and DIC in human plasma. A mixture of petroleum ether-chloroform-ethyl acetate-formic acid (7.5:1:2.5:0.25, v/v) was used as the mobile phase. The two drugs were separated at hRF of 39 and 60 for FEB and DIC, respectively. FEB and DIC were quantified by densitometry at their isoabsorptive point (289 nm). FEB calibration plots were linear between 0.1 and 7 µg mL-1 in both methods A and B. In method B, DIC showed linear response in the range of 0.08-8 µg mL-1. Sample preparation was performed by liquid-liquid extraction using diethyl ether. Both methods did not record any interference from plasma matrix, the studied drugs' metabolites or their decomposition products. They were successfully applied for the determination of the studied drugs in healthy male volunteers after oral administration of FEB or FEB/DIC dosage forms. FEB plasma concentration increased significantly when given with DIC. The proposed methods provided very simple, rapid and cheap approaches that might be attractive for the future pharmacokinetic and bioavailability studies of FEB and/or DIC.

Innovative thin-layer chromatographic method combined with fluorescence detection for specific determination of Febuxostat: Application in biological fluids.

A newly developed thin-layer chromatographic (TLC) method coupled with fluorescence detection for specific determination of Febuxostat (FEB) was designed. The proposed method adopts exposure of FEB on a developed TLC plate to hydrochloric acid vapors, resulting in a large enhancement of its weak fluorescence, permitting its specific and sensitive determination in real human plasma and urine after excitation at 345nm on 60 F254 silica gel plates using toluene-ethyl acetate-methanol-glacial acetic acid; (30:10:5:0.1,v/v/v/v) as mobile phase. The retention factor (Rf) value for FEB was 0.33 ± 0.03 with a correlation coefficient of 0.9974 in the concentration range of 2.5-50ng/band. Upon using polynomial regression, the correlation coefficient was greatly improved (0.9999), with detection and quantification limits of 0.55 and 1.67 (ng/band), respectively. The proposed method was validated according to the International Conference of Harmonization and was successfully used for specific and selective determination of FEB in its commercial dosage form without excipient interference. Moreover, the proposed method was extended to efficient determination of the studied drug in real human plasma and urine samples in the presence of its metabolites without any interference, allowing clinical application of the proposed method for direct FEB determination in biological fluids as well as in pharmacokinetics studies and for quality control of the pharmaceutical dosage form without sample pretreatment or exhausting extraction steps.

Salting-Out Assisted Liquid-Liquid Extraction for Quantification of Febuxostat in Plasma Using RP-HPLC and Its Pharmacokinetic Application.

A rapid and sensitive reversed-phase high-performance liquid chromatography (HPLC) method using novel salting-out assisted liquid-liquid extraction technique has been developed for the quantitative determination of febuxostat (FEB), used for the treatment of gout, in rat plasma. The method was validated according to US FDA guideline. Separation was achieved using a Phenomenex Luna-C18 (250 × 4.60 mm, 5 µm) column and mobile phase composed of potassium dihydrogen orthophosphate buffer 25 mM, adjusted to pH 6.8 with triethylamine:methanol in a ratio of 35:65 (v/v) showing retention time 5.56 and 8.86 min for FEB and internal standard, respectively. The optimal salting-out parameters; 1 mL of acetonitrile and 200 µL of 2 M ammonium acetate salt showed extraction recovery >90% for FEB from plasma. This extraction procedure afforded clear samples resulting in convenient and cost-saving procedure and showed good linear relationship (r > 0.9997) between peak area ratio and concentration from 0.3 to 20 µg/mL. The results of pharmacokinetic study showed that absorption profile of spherical agglomerate of FEB compared to marketed formulation was higher indicating greater systemic absorption. In conclusion, the developed SALLE-HPLC method with simple ultraviolet detection offered a number of advantages including good quantitative ability, wide linear range, high recovery, short analysis time as well as low cost.

Modeling and Simulation for Estimating the Influence of Renal Dysfunction on the Hypouricemic Effect of Febuxostat in Hyperuricemic Patients Due to Overproduction or Underexcretion of Uric Acid.

Whether renal dysfunction influences the hypouricemic effect of febuxostat, a xanthine oxidase (XO) inhibitor, in patients with hyperuricemia due to overproduction or underexcretion of uric acid (UA) remains unclear. We aimed to address this question with a modeling and simulation approach. The pharmacokinetics (PK) of febuxostat were analyzed using data from the literature. A kinetic model of UA was retrieved from a previous human study. Renal UA clearance was estimated as a function of creatinine clearance (CLcr) but non-renal UA clearance was assumed constant. A reversible inhibition model for bovine XO was adopted. Integrating these kinetic formulas, we developed a PK-pharmacodynamic (PK-PD) model for estimating the time course of the hypouricemic effect of febuxostat as a function of baseline UA level, febuxostat dose, treatment duration, body weight, and CLcr. Using the Monte Carlo simulation method, we examined the performance of the model by comparing predicted UA levels with those reported in the literature. We also modified the models for application to hyperuricemia due to UA overproduction or underexcretion. Thirty-nine data sets comprising 735 volunteers or patients were retrieved from the literature. A good correlation was observed between the hypouricemic effects of febuxostat estimated by our PK-PD model and those reported in the articles (observed) (r=0.89, p<0.001). The hypouricemic effect was estimated to be augmented in patients with renal dysfunction irrespective of the etiology of hyperuricemia. While validation in clinical studies is needed, the modeling and simulation approach may be useful for individualizing febuxostat doses in patients with various clinical characteristics.

Determination of Febuxostat in Human Plasma Using RP-LC-UV Method.

A simple and sensitive bioanalytical high-performance liquid chromatographic method with ultraviolet detection was developed and validated for the quantification of febuxostat (FEB) in human plasma. Ketoprofen was used as an internal standard. The analytes were extracted from human plasma samples by precipitation with acetonitrile. The reconstituted samples were chromatographed on C18 Bondapack 10 µm, 250 × 4.6 mm, Waters Column (Ireland) by using a mixture of acetonitrile and 0.5% aqueous phosphoric acid (pH 3) (52 : 48, v/v) as the mobile phase. The chromatographic separation was isocratically performed at room temperature at a flow rate of 1.0 mL/min with UV detection at 315 nm. The method exhibited a linear dynamic range over 0.05-5.00 µg/mL FEB in human plasma. The lower limit of quantification was 0.05 µg/mL. The results of the intra- and interday precision and accuracy studies were within the acceptable limits. The validated method was successfully applied for the determination of FEB in human plasma samples for application in bioequivalence studies.

Population Pharmacokinetics and Therapeutic Efficacy of Febuxostat in Patients with Severe Renal Impairment.

The aim of the present study was to determine the influence of severe renal dysfunction (estimated glomerular filtration rate <30 ml/min/1.73 m(2), including hemodialysis) on the pharmacokinetics and therapeutic effects of febuxostat using a population pharmacokinetic analysis. This study recruited patients with hyperuricemia who were initially treated with allopurinol, but were switched to febuxostat, and it consists of 2 sub-studies: a pharmacokinetic study (26 patients) and retrospective efficacy evaluation study (51 patients). The demographic and clinical data of patients were collected from electronic medical records. Plasma febuxostat concentrations were obtained at each hospital visit. Population pharmacokinetic modeling was performed with NONMEM version 7.2. A total of 128 plasma febuxostat concentrations from 26 patients were used in the population pharmacokinetic analysis. The data were best described by a 1-compartment model with first order absorption. Covariate analysis revealed that renal function did not influence the pharmacokinetics of febuxostat, whereas actual body weight significantly influenced apparent clearance and apparent volume of distribution. The retrospective efficacy analysis showed the favorable therapeutic response of febuxostat switched from allopurinol in patients with moderate to severe renal impairment. No serious adverse event associated with febuxostat was observed irrespective of renal function. The population pharmacokinetic analysis and therapeutic analysis of febuxostat revealed that severe renal dysfunction had no influence on the pharmacokinetic parameters of febuxostat. These results suggest that febuxostat is tolerated well by patients with severe renal impairment.

Simultaneous determination of febuxostat and its three active metabolites in human plasma by liquid chromatography-tandem mass spectrometry and its application to a pharmacokinetic study in Chinese healthy volunteers.

A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of febuxostat and its three active metabolites in human plasma was developed using a ZORBAX SB-C18 column (50 mm × 4.6 mm, 5 µm) and an optimized gradient mobile phase consisting of acetonitrile, water and formic acid. Plasma samples were spiked with the internal standard losartan and then pre-treated using one-step protein precipitation with methanol. Mass spectrometric detection was performed by selective reaction monitoring mode via electrospray ionization source operating in positive ionization mode. The method exhibited good linearity over the concentration range of 10-20,000 ng/mL for febuxostat, 1.0-270 ng/mL for 67M-1 and 67M-2, and 0.8-250 ng/mL for 67 M-4, respectively. The intra- and inter-day precisions were less than 14.7% and the accuracy ranged from -4.3% to 5.1%. The method was successfully applied to a clinical pharmacokinetic study of febuxostat in humans after oral administration of a single dose of febuxostat at 40, 80 and 120 mg.