INTRODUCTION:

Febuxostat is chemically 2-(3-cyano-4-isobutoxyphenyl) -4-methyl-1,3-thiazole-5-carboxylic acid. The structure of febuxostat is shown in Fig.1.Febuxostat is a novel, orally administered non - purine drug that is indicated for use in the treatment of chronic gout and hyperuricemia in patients with gout and chronic tophaceous gout^[1-2]. It is a non-purine selective inhibitor of xanthine oxidase works by noncompetitively blocking the molybdenum protein center which is the active site on xanthine oxidase (XO)^[3-4]. Thus lowering urate concentrations in the body. It has minimal effects on other enzymes involved in purine and pyrimidine metabolism, and is metabolized primarily by glucuronide formation and oxidation in the liver^[5-6].

In a study of subjects with renal impairment, the serum urate–lowering effect of febuxostat was unaltered. Febuxostat, at a daily dose of 80 mg or 120 mg, was further effective than allopurinol at the usually used fixed daily dose of 300 mg in reducing serum urate. Gout is the most common form of inflammatory joint disease in men aged >40 years and is commonly characterized by hyperuricaemia and recurrent attacks of acute arthritis. Sustained hyperuricaemia is considered to be the precursor and the underlying cause of gout. The management of hyperuricaemia associated with gout is therefore one of the most important cornerstones in the management of gout. It is a selective inhibitor of XO that has been developed for the treatment of hyperuricaemia and gout, as it was found to have a potent inhibitory activity for XO/xanthine dehydrogenase (XDH) during evaluation of a range of newly synthesized molecules. A literature survey revealed that several methods have been reported for the determination of Febuxostat alone or in combination in dosage forms or in plasma samples. These methods are UV- Spectrophotometry^[7-11], Spectrofluorimetry[12], RP-HPLC [13-19], GC [20], LC/MS [21], LC-MS/MS[22-27] for the determination of Febuxostat in bulk and pharmaceutical dosage forms. However, the requirement of very simple, quick, efficient, precise, time economy and highly reliable analytical RP-HPLC method for routine quality control purpose always necessities to see a novel and improved method. Hence, the present work was aimed to develop a simple, trouble-free, fast, perfect, and sensitive RP-HPLC method with UV detector for the concurrent estimation of Febuxostat in pure form and pharmaceutical formulations.

Fig.1. Chemical structure of febuxostat

MATERIALS AND METHODS:

Chemicals and Reagents:

Drug was received as a gift from Ajanta Lab, Ltd, Mumbai, India. RP-HPLC grade methanol (Merck) was procured from the local market. All other chemicals and reagents employed were of analytical grade and were purchased from S.D. Fine Chemicals, India.

Instrumentation:

A Gradient RP-HPLC3000 Series (Make: Analytical Technologies Ltd) with UV-3000-Mdetector and grace C18 column was used. A Rheodyne injector with a 20μl loop was used for the injection of sample. The RP-HPLC system was equipped with RP-HPLC Workstation for data processing. A C-18 column (250 x 4.6 mm i.d. particle size 5μm) used for separation.

Chromatographic conditions:

Grace C18 (250mm x 4.6ID, Particle size: 5 micron) was used as stationary phase. Methanol: Potassium Di- Hydrogen Phosphate10mM Buffer pH: 6.8 (70:30) used as mobile phase and was filtered before use through 0.22μm membrane filter. A constant flow of 0.8 ml/min was maintained throughout the analysis and the detection was monitored by UV detector at 315 nm. Chromatogram of standard solution containing febuxostat is shown in Fig. 2.

Preparation of mobile phase and stock solution:

Standard stock solution of febuxostat was prepared by transferring 25 mg in to 25 ml volumetric flask. Sufficient amount of mobile phase Methanol: Potassium Di-Hydrogen Phosphate10mM Buffer pH: 6.8 (70:30) was added, sonicated and remaining volume was made up to the mark with mobile phase, which gave the concentration of 1000μg/ml of the drug. Aliquots of standard stock solution were appropriately diluted with mobile phase to obtain concentration 10-50μg/ml.

Fig.2 Standard chromatograph of febuxostat (10 μg/ml)

Preparation of sample solution for tablets assay:

Twenty tablets of Urifix40 were weighed, crushed and mixed in a mortar and pestle. A portion of powder equivalent to the weight of 10 mg was accurately weighed into 100 ml volumetric flasks and 20 ml of mobile phase was added to flask. The volumetric flask was sonicated for 20 min to effect complete dissolution of drug and the solutions were then made up to volume with Acetonitrile, phosphate buffer. Aliquots of the solution were filtered through a 0.45 μm nylon filter and 1 ml of the filtered solution was transferred to a 10 ml A-grade volumetric flask and made up to volume with mobile phase. Likewise sample was further diluted to get required concentration.

Validation of analytical method:

The proposed RP-HPLC method of analysis was validated in accordance of ICH Q2 (R1) guidelines ^[28] for the parameters like system suitability, specificity, linearity, precision, accuracy, and robustness, limit of detection (LOD) and limit of quantization (LOQ).

System suitability:

The chromatographic systems used for analysis must pass system suitability limits before sample analysis can commence. Set up the chromatographic system allow the RP-HPLC system to stabilize for 40 minutes. Inject blank preparation (single injection) and standard preparation (six replicates) and record the chromatograms to evaluate the system suitability parameters like resolution (NLT 2.0), tailing factor (NMT 1.5), theoretical plate count (NLT 3000) and % RSD for peak area of six replicate injections of Febuxostat standard (% RSD NMT 2.0).The parameters such as tailing factor, % RSD and theoretical plates were studied and found satisfactory. The system suitability data and the optimum chromatographic conditions are reported in Table 1.

Table 1. The system suitability data and the optimum chromatographic conditions.

Parameter	Chromatographic conditions
Instrument	Gradient RP-HPLC 3000 Series (Make: Analytical Technologies Ltd)
Column	Grace C18 (250mm x 4.6ID, Particle size: 5 micron)
Detector	UV-3000-M detector
Diluents	Methanol: Potassium –Di- Hydrogen Phosphate10mM Buffer pH:6.8 (70:30)
Mobile phase	Methanol: Potassium –Di- Hydrogen Phosphate10mM Buffer pH:6.8 (70:30)
Flow rate	0.8 ml/min
Detection wave length	UV at 315 nm.
Run time	10 minutes
Temperature	Ambient temperature (25^oC)
Volume of injection loop	20 µL
Retention time (tR)	7.09 min
Theoretical plates [th.pl] (Efficiency)	6831
Tailing factor (asymmetry)	1.08

Linearity:

Under proposed experimental conditions, the relationship between the area and concentration of Febuxostat was studied. Linearity was checked by preparing standard solutions at 5 different concentration levels of each of Febuxostat. Standard solutions (10, 20, 30, 40, 50µg/mL) of Febuxostat were injected into the RP-HPLC system to get the chromatograms. The average peak area and retention time were recorded. The calibration curve was constructed between concentrations versus peak area by the prepared concentration of 10-50 µg/mL of stock solution. The linearity range was found to be 10-50 µg/mL and the calibration graph of febuxostat shown in Fig.3.Results show that a phenomenal correlation exists between peak area and concentration of drug within the linearity range. The Summary of validation parameters are shown in Table 2.

Table 2. Summary of validation parameters.

Parameter	Result
Linearity range (µg/mL)	10-50 µg/mL
Liner Regression equation	y = 120913x – 186788
R²	0.9962
Intraday precision (% RSD)	0.130 %
Interday precision (% RSD)	0.350 %
Recovery	98.68 - 99.38 %
LOD (µg/mL)	0.247
LOQ (µg/mL)	0.750
Robustness	Robust

Fig. 3 Calibration curve of febuxostat.

Precision:

Precision of the method was evaluated by determining intra-day precision and inter-day precision and express in terms of % RSD (% relative standard deviation). The repeatability was studied by repeating the assay three times in the same day and intermediate precision was studied by repeating the assay on three different days, three times on each day. The intraday and inter-day variation for determination of Febuxostat was carried out at three different concentration levels 10, 30, 50 µg/ml). The results of intraday and interday precision are shown in Table 3.

Table 3. Interday and intraday precision study of febuxostat.

Concentration µg/mL	Peak area	Standard deviation	% Relative Standard deviation
Interday precision (n=3)
10	908809	0.197	0.350
30	3269480	0.158	0.156
50	5987904	0.159	0.175
Intraday precision (n=3)
10	910004	0.157	0.135
30	3272480	0.198	0.116
50	5997904	0.179	0.165

Recovery:

Accuracy is the measure of how close the experimental value is to the true value. Recovery studies by the standard addition method were performed with a view to justify the accuracy of the proposed method. Previously analyzed samples of febuxostat (12μg/ml) were spiked with 50, 100, and 150% extra febuxostat standard and the mixtures were analyzed by the proposed method. The experiment was performed in triplicate. The results of recovery study are presented in Table 4.

Table 4. Recovery study of febuxostat.

Sr. no.	% level of recovery	Amount of drug added (μg/mL)	*Mean (± SD) amount found (μg/ml)**	% Recovery
1	50%	50	149.47 ± 0.74	99.64
2	100%	100	198.07 ± 0.18	99.03
3	150%	150	246.77 ± 0.44	98.70

Table 5 Robustness results of Febuxostat.

Sr. No	Parameter	Optimized	Used	Retention time (tR), min	Plate count$	Peak asymmetry#	Remark
1	Flow rate (±0.2 mL/min)	0.8 ml/min	0.8 ml/min	6.98	6831	1.08	Robust
			1.0 ml/min	6.78	6841	1.07	Robust
			1.2 ml/min	6.94	6823	1.08	Robust
2	Detection wavelength (±5 nm)	315 nm	310	6.98	6832	1.05	Robust
			315	6.78	6840	1.09	Robust
			320	6.94	6825	1.07	Robust
3	Mobile phase composition (Methanol: Phosphate buffer pH:6.8)	(70:30)	70:25	6.97	6838	1.08	Robust
			70:30	6.84	6837	1.09	Robust
			70:35	6.91	6830	1.08	Robust

ROBUSTNESS:

Robustness of the method is its ability to remain unaffected by small changes in variety of parameters such as the slight variation in acetonitrile percentage composition of the mobile phase, flow rate, detection wavelength. The result of robustness study is shown in Table 5indicated that the small change in the conditions did not significantly affect the determination of Febuxostat.

LOD and LOQ:

Limit of detection is the lowest concentration in a sample that can be detected, but not necessarily quantified under the stated experimental conditions. The limit of quantitation is the lowest concentration of analyte in a sample that can be determined with acceptable precision and accuracy.

Limit of detection and limit of quantitation were calculated using following formula LOD = 3.3 σ/S and LOQ = 10 σ/S, where, σ is the standard deviation of response and S is the slope of the calibration curve. The LOD and LOQ values are presented in Table 2. The results of LOD and LOQ supported the sensitivity of the developed method.

Assay of marketed formulations:

Using the developed RP-HPLC chromatographic method, assay of Febuxostat in tablet was carried out as mention in the experimental section. Six replicate determinations were made. Satisfactory results were obtained and were good agreement with the label claim and assay results were shown in Table 6. The results were very close to the labeled value of commercial tablets.

Table 6 Assay of marketed formulations of Febuxostat.

Sr. No	Brand name	Labelled claim	Amount found	% assay
1	Febucip	40 mg/tablet	39.67 mg/tablet	99.17± 0.97
2	Urifix 40	40 mg/tablet	39.56 mg/tablet	98.90± 0.34

RESULT AND DISCUSSION:

The present study was aimed at developing a precise, sensitive, rapid and accurate RP-HPLC method for the analysis of Febuxostat in bulk drug and in pharmaceutical dosage forms. In order to achieve extraordinary retention time and peak asymmetry, C18 stationary phase column (250 mm X 4.6 mm i.d., 5 μm particle size) and mobile phase composed of methanol: potassium Di- Hydrogen Phosphate10mM Buffer pH: 6.8 (70:30)at a flow rate of 0.8 mL/min was selected. The retention time for Febuxostat was found to be 6.98 minute. The correlation coefficient (0.9999) of regression was found almost equal to one in the range of 10-50 μg/mL which states that the method was good linear to the concentration versus peak area responses. The comparison of chromatograms of placebo, standard and sample, there was no interference observed from the peaks of placebo, standard and sample. It shows that the method is specific. The precision studies were performed and the % RSD of the determinations was found to be 0.13 for intra-day precision and 0.34 for inter-day precision which are within the limits which indicates that the proposed method was found to be precise. The accuracy of the method was found to be good with the overall % RSD for recovery at 10 %, 30 % and 50 % levels were all within the limits which indicate that the proposed method was found to be accurate. Method validation following ICH guidelines indicated that the developed method had high sensitivity with LOD of 0.247 μg/mL and LOQ of 0.750 μg/mL. The method was found to be robust even though on slight deliberate variation in the method conditions did have a tiny effect on the peak asymmetry, plate count and retention time and all are within the limits which indicated that the method is robust. Range is the minimum and maximum concentration of the sample at which the analytical procedure gives reproducible results. Range can be determined by linearity, accuracy and precision studies. The retention time of the sample solution of Febuxostat tablet was found to be 6.98 minutes, which is similar to that of the standard solution of Febuxostat.

This indicates that there is no drug-excipient interference and the drug is decorously resolved by the developed method. Robustness determines the reproducibility of the test result with small and deliberate variations in the method parameters. The effectiveness of the deliberate little variations was observed on the flow rate, wavelength and mobile phase composition. The results of robustness study also indicated that the method is robust and is unaffected by small deliberate variations in the method parameters. The proposed method is simple, fast, accurate, and precise and can be used for routine analysis in quality control of febuxostat.

CONCLUSION:

The developed RP-HPLC method provides a convenient and efficient method for the estimation of Febuxostat in dosage form. This method has various advantages like less retention time, low solvent consumption, outstanding peak symmetry, and phenomenal linearity, highly sensitive, precise, accurate and robust. The mobile phase can be easily prepared and diluents are economical and readily available and it does not need sample preparation with sophisticated techniques. The drug solutions employed in the study were stable up to 48 hours. These attribute the high quality of the method. There was no interference from the excipients used in the tablet formulation. The proposed method can be used for the routine analysis of Febuxostat in pharmaceutical dosage forms for routine application in quality control laboratories.

ACKNOWLEDGEMENT:

The authors are thankful to the management of MGV College of Pharmacy, Panchvati, Nashik for providing the, necessary facilities to carry out this research work and to Precise Chemipharma Pvt. Ltd, Dindori, Nashik for providing gift sample of pure drug.

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Received on 12.03.2017 Modified on 18.05.2017

Asian J. Research Chem. 2017; 10(6):713-718.

DOI: 10.5958/0974-4150.2017.00121.3