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

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


Introduction
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][6][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][9][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][12][13][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).

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 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.

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 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

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. 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.

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.

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  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.
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.

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 10 6 LUX hours), UV light for a total exposure of 200 W·hr/m 2 , 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       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.