Research Article - (2015) Volume 6, Issue 10

Development and Validation of Reverse Phase-Ultra Performance Liquid Chromatographic Method for Estimation of Related Substances in Febuxostat Drug Substance

Maheswara Reddy Musirike*, Hussain Reddy K and Useni Reddy Mallu
Department of Chemistry, Sri Krishnadevaraya University, Ananthapur, Andhra Pradesh- 515003, India
*Corresponding Author: Maheswara Reddy Musirike, Department of Chemistry, Sri Krishnadevaraya University, Kadiri- Ananthapur Hwy, Kandukuru, Anantapur, Andhra Pradesh 515591, India, Tel: 085542 55700 Email:


A novel high speed, high resolution Reverse phase-UPLC method was developed for the quantification of related substances in Febuxostat drug substance. The separation of drug from the possible impurities was achieved on a Halo C18 column. The innovative approach of using stationary phase with sub 2 μ particles provides a comprehensive combination of selectivity and speed. 10 mM mono basic potassium phosphate buffer at pH 2.7 and acetonitrile mixture was selected as mobile phase. Flow rate and detection were kept at 0.8 mL/min and 320 nm respectively. The developed UPLC method was subjected to validation parameters. System precision, accuracy, specificity, limit of detection, limit of quantification and linearity were established as per the guidelines recommended by ICH. Stability indicating nature of the method was also performed by exposing the sample under various conditions like acid, base, peroxide and photo stability exposures. Total analysis run time 7.0 minutes indicates the speed and cost saving initiation of the developed method. Using the method one can carry out the quantitative estimation of related substances in Febuxostat drug substance, further the same method can be adopted for determination of drug substance assay also.

Keywords: Febuxostat; Ultra performance liquid chromatography; Hyperuricemia with gout; Related compounds; Xanthine oxidase inhibitor


Febuxostat is a xanthine oxidase inhibitor indicated for the chronic management of hyperuricemia in patients with gout [1]. The recommended starting dose of drug is 40 mg once daily. For patients who do not achieve a serum uric acid less than 6 mg per dL after 2 weeks with 40 mg, Uloric 80 mg is recommended. The primary mode of action of a xanthine oxidase inhibitor, achieves its therapeutic effect by decreasing serum uric acid.The objective of this work was to develop a cost effective ultra fast reverse phase UPLC method, the developed method was validated as per regulatory guidelines and transfer the method to quality control lab for analysis of related substances in Febuxostat. As per the literature Febuxostat Determination was done by spectrophotometric method [2] and RP-HPLC [3,4] methods. LC-MS/MS [5-7] assay method was proposed for bioequivalence and pharmacokinetics evaluation. Since this drug is being marketed in domestic and international market the present investigation by the author was to develop a rapid, accurate and precise RP-UPLC method [8-10] for the determination of related substances. The aim of this paper is to develop a cost effective and fast method. The run time for the newly developed method was 7.0 minutes. The innovative approach of using stationary phase with sub 2 μ particles [11-14] provides a comprehensive combination of selectivity and speed. The validation parameters [15,16] provide valuable information on precision accuracy, limit of detection, limit of quantification and linearity of related substances. The method was subjected to validation according to ICH requirements [17,18] (Figure 1).


Figure 1: Chemical structures of febuxostat and its related substances.

Materials and Methods

Instrumentation and reagents

Test samples and reference standards of Febuxostat were donated by Apotex India pvt ltd. Acetonitrile was purchased from Ultra Scan Ltd. Ultra performance liquid chromatography from Waters with gradient elution. MilliQ purification system was used to get HPLC grade water. Halo C18, 100 x 2.1 mm 2.7 μm column purchased from advanced materials technology.

Chromatographic conditions

The chromatographic separation was achieved on a Halo C18, 100 x 2.1 mm 2.7 μm Column. Mobile phase consists of 10 mM monobasic phosphate buffer with 0.2% Triethyl amine and pH of the solution was adjusted to 2.7. HPLC grade acetonitrile was used as organic modifier. Mobile phase flow rate was kept at 0.8 mL/min. Gradient program was set as Time/% of solution B: 0/40, 3/40, 3.5/70, 4/75, 5/80, 5.5/40, 7/40. Column temperature was maintained at 50°C and detection was carried at 320 nm. Sample compartment was maintained at 10°C with an injection volume of 1 μL.

Preparation of standard and sample

A mixture of standard solution was prepared by weighing febuxostat and its related compounds to yield a final concentration of 0.10% of febuxostat and 0.15% of each Impurity A, Impurity B, Impurity C and Impurity D with respect to the sample concentration of 0.5 mg/ml. Buffer and acetonitrile in the ratio 1:4 was used as the diluent for preparation of sample and standard solution.

Method validation

Specificity: Specificity is the ability of method to measure the analyte response in the presence of its potential impurities. The specificity of the developed RP-UPLC method was demonstrated in presence of sample diluent and its four potential impurities.

System suitability: To ensure system suitability, a standard solution was injected on to the system and verified spectral purity of individual peaks to ensure that no co elution has occurred. Tailing factor (T) column efficiency (N) and resolution (R) were calculated for febuxostat and its related substances.

Precision: Precision of the method was reported by injecting six replicates of standard solution consecutively under the same analytical conditions. The %RSD of individual peaks was calculated. Intermediate precision of the method was also evaluated using different analyst, different day and different make of instrument in the same laboratory.

Limit of detection (LOD) and limit of quantification (LOQ): The LOD and LOQ for Febuxostat and its related compounds were determined by injecting series of diluted impurity standard solution to a level such that % RSD was not more than 10% at LOQ level. Precision carried out at LOQ level by injecting six individual preparations and calculated the % RSD.

Linearity: Linearity for the related substances method was prepared by serially diluting the impurity stock solution to required concentration levels. The solutions were prepared at five different concentration levels ranging from LOQ to 160% with respect to specification limits. The calibration curve was drawn by plotting the peak areas of impurities versus its corresponding concentrations. Correlation coefficient of the calibration curve, slope and relative response factors were reported.

Accuracy: Febuxostat sample solution was spiked with impurity standard solutions at three concentration levels corresponding to LOQ, 100% and 160% of impurity concentration. The % recovery was calculated.

Results and Discussion

Method development and optimization

Febuxostat and related substances are closely related structures so the main target of the method was to have more specific method to achieve the separation between all impurities from the drug substance effectively and to support routine quality check at competitive time period. The wavelength of detection was selected at 320 nm as all the related impurities and febuxostat shown maximum absorbance at selected wavelength. Resolution and peak symmetry were good in Halo C18. A simple gradient was selected to resolve all the identified impurities in the standard solution and to eliminate interference with diluent and unidentified peaks from sample.

System suitability results

The peak shape of febuxostat drug substance was found symmetric and well separated by its potential process impurities. A typical system suitability chromatogram of sample diluent, standard solution and test solution chromatograms are shown in Figure 2a-2c.


Figure 2: A typical chromatogram showing the febuxostat (a) sample diluent (b) Standard solution

In the optimized conditions, febuxostat and its related substances were well resolved with a resolution of more than 2.0.The tailing factor is in the range of 1.0-1.2 which indicates symmetry of peaks. Theoretical plates more than 10000 show the efficiency of the column. System suitability parameters for febuxostat and its related substances are tabulated in Table 1.

Component RT Resolution Tailing factor Plate count Peak angle Peak threshold
Impurity A 1.451 - 1.2 10800 3.118 5.232
Febuxostat 2.656 15.8 1.0 13293 5.523 9.151
Impurity B 3.430 9.2 1.0 48116 4.140 6.438
Impurity D 4.430 20.3 1.2 387969 3.243 3.632
Impurity C 4.556 4.1 1.1 341520 2.903 4.296

Table 1: System suitability results.

Method validation results

Precision: System precision was evaluated by performing six replicate injections for the standard solution at specification level. The % relative standard deviation of 6 injections was within the acceptable limit.

The obtained results are in the range of 1.4-3.2 which indicates the precision of the system to proceed for analysis. Results are tabulated in Table 2.

Inj.# Febuxostat Impurity A Impurity B Impurity C Impurity D Criteria
1 2811 3659 3386 3195 3367 NMT 5%
2 2933 3503 3431 3089 3443
3 2957 3538 3311 3142 3591
4 3075 3601 3211 3091 3467
5 2865 3340 3449 3131 3380
6 2964 3463 3281 3171 3348
Mean 2934 3517 3345 3136 3433
SD 90.93 111.56 92.71 42.51 90.33
%RSD 3.1 3.2 2.8 1.4 2.6

Table 2: System precision results.

Limit of detection (LOD) and limit of quantification (LOQ): Data obtained for limit of detection (LOD) and limit of quantification (LOQ) of febuxostat drug substance and its related substances are tabulated in Table 3. Sensitivity is the ability of method to detect and quantify the impurities present in the sample accurately. %RSD for area count of 6 consecutive injections should not be more than 10.0% at LOQ level. The Accuracy at LOQ level should be in the range of 80 to 120%. The Precision at LOQ level and accuracy at LOQ level were found to be within the specified limits.

Inj.# Febuxostat Impurity A Impurity B Impurity C Impurity D
 LOQ(µg/ml) 0.404 0.426 0.420 0.424 0.428
LOD(µg/ml) 0.13 0.142 0.140 0.141 0.142
LOQ-Limit of Quantitation; LOD-Limit of Detection

Table 3: Limit of quantitaion and detection.

Linearity: Linearity of the method is to establish a linear relationship of concentration against response. Solutions of febuxostat and its related substances are prepared from LOQ level to 160% of the specification limit. The correlation coefficient obtained was greater than 0.99. The regression statistics for febuxostat drug substance and its related substances are tabulated in Table 4.

Level Febuxostat Impurity A Impurity B Impurity C Impurity D
Conc.(µg/mL) Area (µV*
Conc.(µg/mL) Area (µV*
Conc.(µg/mL) Area (µV*
Conc.(µg/mL) Area (µV*
Conc.(µg/mL) Area (µV*
LOQ 0.4040 1237 0.4256 1310 0.4200 1281 0.4240 1197 0.4280 1365
80% 0.8080 2453 0.8512 2692 0.8400 2708 0.8480 2360 0.8560 2744
100% 1.0100 3143 1.0640 3712 1.0500 3532 1.0600 3185 1.0700 3383
120% 1.2120 3576 1.2768 4270 1.2600 4242 1.2720 3940 1.2840 4027
160% 1.6160 4749 1.7024 5524 1.6800 5296 1.6960 5073 1.7120 5437
Slope 2886 3341 3233 3115 3154
R2 0.9990 0.9960 0.9960 0.9980 1.0000
RRF 1.00 0.86 0.89 0.93 0.92
RRF-Relative Response factor, R2- Correlation Coefficient

Table 4: Linearity results.

The result shows that an excellent correlation existed between the peak response and concentration of the analyte and impurities. Relative response factor is established to estimate the factor of impurity response against drug substance response to calculate the content of impurity present in the drug substance.

Accuracy: Accuracy of the method can be determined by spiking known concentrations of standard solution to the sample. The obtained recovery value indicates the trueness of the method to estimate impurities. Related substances of febuxostat spiked to the sample over a concentration range varying from QL to 160% of their respective target analyte concentrations. The Acceptance criteria for the accuracy are 80% to 120%.

The obtained percentage recovery value of related substances is in the range of 95.2 % to 102.3% which declares the method accuracy. Accuracy results are reported in Table 5.

S.NO Accuracy Level Impurity A Impurity B Impurity C Impurity D Criteria
1 LOQ 102.4% 87.5% 105.2% 101.3%   80% to 120%
2  100% 100.5% 85.6% 103.1% 100.0%
3  160% 101.8% 95.2% 102.3% 99.4%

Table 5: Accuracy results.

Forced degradation study: Degradation studies were performed to demonstrate stability indicating nature of the method. Febuxostat test sample was exposed to various stress conditions like heat and humidity (40°C and 70% RH for 7 days), thermal (60°C for 7 days) and photolytic conditions of fluorescent light (1.2 x 106 LUX hours), UV light for a total exposure of 200 W·hr/m2, acid hydrolysis (0.1 N HCl 80°C for 24 Hrs), base hydrolysis (0.1 N NaOH, 80°C for 24 Hrs) and oxidative stress. Testing of peak purity concludes the homogeneity and interference of unidentified impurities with peak of interest. The obtained peak purity value gives a clear indication of separation between stressed impurities with related substances of febuxostat.

Peak obtained in all the stress conditions was homogenous and unaffected by the presence of its degradation impurities, confirming the stability indicating nature of the method. Mass balance also established to match up the sum of impurities with its assay value against reference unstressed sample. The results from forced degradation studies are summarized in Table 6. Figure 3a-3c indicates stressed sample chromatograms under acidic, basic and peroxide conditions.

Stress condition Conc.µg/mL Match angle Match threshold Purity angle Purity Threshold Mass balance
Non stressed 504 2.880 10.550 0.075 10.123 100
Acid hydrolysis 520 2.542 10.235 0.012 10.225 99.8
Base hydrolysis 510 2.325 10.835 0.085 10.522 98.9
Oxidation 530 1.987 11.123 0.023 10.995 99.9
Heat and humidity 525 1.897 11.556 0.093 10.789 99.9
Photo stability 515 2.552 11.005 0.075 10.123 99.9
Dry heat 518 2.342 11.123 0.092 10.889 99.8

Table 6: Forced degradation studies.


Figure 3: A typical chromatogram showing the febuxostat (a) Acid hydrolysis (b) Base Hydrolysis (c) Oxidative degradation.


A fast Reverse phase-UPLC method was developed for the estimation of related substances in Febuxostat drug substance. The developed method was subjected to method validation parameters as recommended by ICH. Stability indicating nature of the method is also established by assessing forced degradation studies. The shorter run time demonstrates that the method is cost effective, time effective and even uses lesser quantity of solvents aiming towards green chemistry. The developed method was specific, precise, accurate and linear to estimate accurate amount of impurities present in the sample. Degradation studies confirmed the homogeneity and free of interferences with the peak of interest.

The method can be adopted to determine the related substances of drug substance in quality control labs. The same procedure can also be used to perform assay of drug substance.


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Citation: Musirike MR, Hussain Reddy K, Mallu UR (2015) Development and Validation of Reverse Phase-Ultra Performance Liquid Chromatographic Method for Estimation of Related Substances in Febuxostat Drug Substance. Pharm Anal Acta 6:431.

Copyright: © 2015 Musirike MR, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.