Development and Validation of An Analytical Liquid Chromatography-Tandem Mass Spectroscopy Method for the Estimation Febuxostat in Pharmaceutical Formulation


Lingamallu Venkata Sai Krishna1, Narenderan. S. T1, Ramshankar Nayak1,  Meyyanathan. S. N*1, Babu. B1, Kalaivani. M2

1Department of Pharmaceutical Analysis, JSS College of Pharmacy (JSS Academy of Higher Education and Research, Mysuru) Udhagamandalam, India.

2Indian Pharmacopoeia Commission, New Delhi.

*Corresponding Author E-mail:



A rapid LC-MS/MS method has been developed and validated for the quantitative determination of Febuxostat from the commercially available formulations. The separation was achieved using Zorbax SB C18 column (4.6 × 50 mm, 5 μm) as a stationary phase and the mobile phase consists of (10 mM) Ammonium formate (pH4.0): methanol (10:90 v/v) with a flow of 0.6 mL/min. Detection was carried out by triple quadrupole mass spectrometry with electrospray ionization in positive mode with proton adducts at m/z 316.95260.95 to monitor Febuxostat within a run time of 2 min. The linearity of the method was found over a concentration range of 4 to 55 ng/mL with a regression analysis of 0.997. The percentage recovery of the present method was found to be 97.16 to 99.46%. The LC-MS/MS method was validated as per ICH guidelines. The developed method can be successfully applied for the estimation of Febuxostat in the commercial formulation and in bulk drug.


KEYWORDS: Febuxostat, Formulation, LC-MS/MS, Validation, Electrospray ionization.




Febuxostat chemical name is 2-[3-cyano-4-(2-methylpropoxy) phenyl]-4methylthiazole-5-carboxylic acid is non-purine, a selective inhibitor of Xanthine Oxidase (XO) that prevents the normal oxidation of purines to uric acid. Febuxostat is commercially available as tablets (40 and 80 mg)1. It is available as a white crystalline powder which is freely soluble in dimethylformamide, soluble in dimethyl sulfoxide, sparingly soluble in ethanol, slightly soluble in methanol and acetonitrile, practically insoluble in water. Febuxostat is used in the treatment of chronic Gout and hyperuricemia.


The literature survey revealed an analytical method for Febuxostat was estimated by Spectrophotometry2,3, HPLC4-6 and to best of our knowledge, liquid chromatography-tandem mass spectroscopy has been previously reported in human plasma7-8. The aim of the current study was to develop a highly sensitive method for the estimation of Febuxostat in bulk and formulations and validate as per ICH guidelines.




Working standard of Febuxostat was provided as a gift sample from Indian Pharmacopoeia Commission, New Delhi, India. Methanol of LC-MS/MS grade by Sigma Aldrich, Methanol by Rankem Fine Chemical Limited and Water of LC-MS/ MS grade from Milli-Q RO system (Millipore, Bedford, USA) were used.


Equipment and chromatographic conditions9:

LC system coupled with tandem Quadrupole mass spectrometry (Shimadzu 8030, Tokyo Japan) equipped with electrospray ionization (ESI) interface, LC-20AD pump, SPD-M20 PDA detector, CTO-20AC column oven, CBM-20 Alite controller and SIL-20AC Auto-sampler was used. The data were recorded using Lab solution data station software. Isocratic separation was achieved using Zorbax SB C18 column (4.6 × 50 mm, 5 μm) as a stationary phase and the mobile phase consists of (10 mM) Ammonium format (pH4.0): methanol (10:90 v/v) with a flow of 0.6 mL/min and injection volume of 10μL was employed.


Selection of a mass range:

A1000 ng/mL of Febuxostat was infused into the mass spectrometer directly and the conditions for the operation were optimized. Obtained transitions were 316.95260.95 and 316.9573.0 m/z was used to monitor Febuxostat (Fig. 1).



Fig. 1: Typical Mass scan spectra of Febuxostat


Sample preparation:

Preparation of working standard solution:

Febuxostat was dissolving in 10 mL of Methanol to produce a concentration of 1 mg/mL. The stock solution was refrigerated at 2-8°C and stored. Further, the working solutions were obtained by diluting the stock solution with diluent methanol.


Method validation:

Validation of the method for specificity, linearity, accuracy, precision, range, Quantitation limit, and detection limit, robustness, and system suitability as per the ICH guidelines10.



The analyte response measurement in the presence of other drugs, excipients, and their potential impurities can be termed as specificity.





The average of six determinations at seven concentration levels covering the range of 4-55ng/mL for Febuxostat, the evaluation of linearity was performed. Calculation of the coefficient correlation, slope and intercept values was done by using a calibration curve for linearity evaluation.



The accuracy of the method was determined by recovery studies according to ICH guidelines. The pre-analyzed samples were spiked with standard drug Febuxostat.



Evaluation of precision was carried out by inter-day and intra-day precision. Study samples consisted of three concentration levels (six replicates) of low (LQC), medium (MQC) and high (HQC) quality controls, i.e. 4, 28, 50ng/mL respectively. The report used for precision was from the regressed concentration of the percent relative standard deviation (%RSD).


Limit of detection (LOD) and limit of quantification (LOQ):

Determination of LOD and LOQ was by the signal-to-noise ratio. LOD ratio was 3:1 whereas LOQ, the drug could be quantified with minimum peak area in the ratio of 10:1.



The alteration in the condition of the experiment like operators, the source of reagents, similar type column and optimized conditions like pH, mobile phase ratio, and flow rate were studied for the robustness of the method.


System suitability:

For method development, the test for system suitability is an integral part. Three replicates injections of the sample solution were evaluated for retention time (RT), a number of theoretical plates (N) and Tailing factor (T).




To determine that the excipients used are not interfering with the main compound peak, test for specificity needs to be done. No peaks were eluted along with the retention time (Fig. 2). Hence, the developed method results showed that it was selective for the determination of Febuxostat in the formulation.



Fig. 2: Typical standard chromatogram of Febuxostat with blank


Calibration curve:

The linearity was evaluated by six determinations at seven concentration levels with a range of 4-55ng/mL for Febuxostat and the standard deviation (SD) were found to be within the limits. A calibration curve was found to be linear with a mean regression of equation (y = 2126.2x - 637.59, R² = 0.9978, S.D. = 0.54) respectively.



The accuracy of the method was carried out for three quality control (LQC, MQC, and HQC) samples by standard addition method, and the accuracy was found to be 93.34 to 98.86 %. Application of the developed method for the estimation of a commercial formulation of Febuxostat.



The precision of the method was determined by the intra-day and inter-day precision studies at three different concentrations and they were found to be within the limits (Table 1).


Limit of detection and limit of quantification:

The lowest limit detected for the method for Febuxostat was at1.5 ng/mL based on the signal-to-noise ratio of 3:1. Due to the increase in the sensitivity of the method, quantification was done at 4 ng/mL for Febuxostat.



The robustness of the method was determined by alteration of the chromatographic conditions and the results obtained were found to be within the limits proving that the developed method was found to be robust.


Application in Formulation:

Twenty tablets were taken, weighed accurately and powdered. From the powdered sample, the amount equivalent to 100 mg of Febuxostat was accurately weighed and transferred to 100 ml volumetric flask. About 50 ml of methanol was added and the solution was sonicated for about 30 min and then the volume was made up to 100 ml using methanol. Further, the solution was shaken thoroughly and filtered through a syringe filter and the quality control samples of low-quality control (LQC) 4 ng/ml, medium quality control (MQC) 28 ng/ml and high-quality control (HQC) 50 ng/ml were prepared. Estimated results have been shown in Table 2 and Fig. 3.


Table 1: Accuracy and precision results of Febuxostat



Amount found (ng/mL) ± SD*



Accuracy (% N)

Precision (% RSD)**

Accuracy (% N)

Precision (% RSD)


3.7 ± 0.05






27.3 ± 0.57






49.4 ± 0.40





*SD: Standard Deviation, RSD: Relative Standard Deviation.

Table 2: Recovery results for Febuxostat in the formulation


Label claim

Amount taken for assay (ng/mL)

Amount Found ± SD

Recovery %


Febuget 80


80 mg


3.8 ± 0.08



27.6 ± 0.15



49.7 ± 0.20


*SD: Standard Deviation

Fig. 3: Typical sample chromatogram of Febuxostat in the formulation



A novel simple, precise, accurate and validated, the liquid chromatography-tandem mass spectroscopy method has been developed and validated. The developed method can be applied for the estimation of Febuxostat in the commercial formulation and in bulk drug.



The authors declare that there are no conflicts of interest.



The authors are grateful to the Indian Pharmacopoeia Commission, New Delhi for providing the standard Febuxostat as a gift sample.



1.       Drug details, URL:

2.       Rajyalakshmi Ch., Benjamin T., Ram Babu C. Development and Validation of Zero and First Order Derivative UV-Spectrophotometric Method for Determination of Febuxostat in Bulk and in Formulation. Research. J. Pharm. and Tech. 2013; 6(2): 208-211.

3.       J. M. Derasari, V. B. Patel. Spectrophotometric Determination of Febuxostat and Diclofenac Potassium in Their Combined Dosage Form by Simultaneous Equation and First Order Derivative Methods. Asian J. Research Chem. 2013; 6(10): Page 968-972.

4.       D. M. Darandale, K.B. Erande, S.R. Tambe, R.S. Bhamber. Development and Validation of RP-HPLC Method for the Determination of Febuxostat in Bulk and Pharmaceutical Dosage Form. Asian J. Research Chem. 2017; 10(6):713-718.

5.       Muvvala SS, Ratnakaram VN, Nadendla RR. Validated RP-HPLC method for the estimation of febuxostat in bulk drugs. Int J pharmTech Res. 2012; 4(4): 1358-1366.

6.       Kumar RS, Ravikumar BVV, Anjaya Kumar P. A RP-HPLC method development and validation for estimation of febuxostat bulk and pharmaceutical dosage forms. Int Res J Pharm. 2012; 3(11): 205-208.

7.       Chandu BR, Kanala K, Hwisa NT, Katakam P, Khagga M. Bio-equivalance and pharmacokinetic  study of febuxostat in human plasma by using LC-MS/MS with liquid liquid extraction method. Spinger plus, 2013; 2(194): 1-10.

8.       Pal N, Rao AS, Kumar PR. New method development and validation for the determination of febuxostat in human plasma by liquid chromatography –mass spectrometry. Int J Pharm Pharm Sci. 2016; 8(9): 61-70.

9.       Mitesh D Phale, Dipti Korgaonkar. Current Advance Analytical Techniques: A Review. Asian J. Research Chem. 2009; 2(3): 235-238.

10.     ICH validation, URL: Q2_R1/Step4/Q2_R1__Guideline.pdf.







Received on 20.11.2018          Modified on 29.12.2018

Accepted on 17.01.2019        © RJPT All right reserved

Research J. Pharm. and Tech. 2019; 12(5):2137-2140.

DOI: 10.5958/0974-360X.2019.00354.8