A New Isocratic RP-HPLC Assay Method Development and Validation of Febuxostat in Tablet Dosage Form

 

Avinash Mahadeo Bhagwat*, Pratiksha Vitthal Awaghade, Atisha Jayprakash Katte, Nishigandha Dayanand Naikawadi

Department of B. Pharmacy, YSPM’s Yashoda Technical Campus, Faculty of Pharmacy, Wadhe, Satara 415 011

*Corresponding Author E-mail: avinashbhagwat2167@gmail.com

 

 

INTRODUCTION:

Febuxostat used to treat hyperuricemia and persistent gout. More effective than conventional allopurinol doses, but not as effective as larger allopurinol doses. Febuxostat works by reducing the production of uric acid in the body. It's used to keep gout attacks at bay. Febuxostat is a non-hygroscopic powder that is readily soluble in dimethyl formamide, soluble in dimethyl-sulfoxide, marginally soluble in methanol and acetonitrile, and practically insoluble in water. The melting temperature ranges from 205°C to 208°C. 2-[3-cyano-4-(2- methylpropoxy) phenyl]-4-methyl-1, 3-thiazole-5-carboxylic acid is the chemical name for Febuxostat. C16H16N2O3S is its chemical formula, and its molecular weight is 316.375g/mol.

 

 

Fig: Structure of Febuxostat

 

High-performance liquid chromatography (HPLC) is an important quantitative and qualitative analysis technique. It's a tool for separating things. In biochemistry and analytical chemistry, the distribution of the analyte (sample) between mobile and stationary phases is used to identify and quantify each component in a combination. HPLC analysis is exact, specific, and precise. HPLC is an essential tool for drug analysis in the pharmaceutical industry. HPLC stands for high-performance liquid chromatography. The pressurized liquid including a solvent and sample mixture is driven through a packed column by the pump. There is a stationary phase and a movable phase in this system. Any drug analysis should use high-performance liquid chromatography (HPLC). Confirm the drugs identify and provide qualitative and quantitative data.¹

 

Assay of Febuxostat by RP-HPLC^2,3

Phosphate Buffer (pH 3.0):

4.08gm of Dihydrogen Orthophosphate dissolved in 1000ml of purified water. Adjust the pH to 3.0 with Orthophosphoric acid.

 

Mobile Phase:

Phosphate Buffer: Methanol: Acetonitrile (40:20:40). Mixed, Sonicated and filtered through 0.45-micron nylon filter paper.

 

Chromatographic Conditions:

Column	150 x 4.6mm, 3.5 µm, Agilent XDB C18column
Detection Wavelength	317nm
Flow Rate	1.0ml / min.
Injection Volume	20µl
Column Oven Temperature	400C
Run time	7min
Mobile Phase	Phosphate Buffer: Methanol: Acetonitrile (40:20:40)

 

Standard Preparation:

Weigh accurately and transfer about 40mg of Febuxostat standard in 100ml volumetric flask. Add about 70% mobile phase and sonicate for 5 minutes. Allow the solution to attend room temperature. Dilute up to mark with Mobile phase. Dilute 10ml of this solution with 100 ml with of Buffer. Crush and make Powder of 20 Tablets at a time to fine powder in mortal. Mix to make uniform and perform assay from it.

 

Sample preparation:

Weigh accurately and transfer sample powder containing about 40mg of Febuxostat in 100ml volumetric flask. Add about 70% mobile phase and sonicate for 20 minutes. Allow the solution to attend room temperature. Dilute up to mark with Mobile phase. Further Dilute 10 ml of this solution to 100ml of volumetric flask. Dilute with mobile phase. Filter through 0.45-micron syringe filter. And inject.

 

System suitability criteria^4,5:

1.     % RSD for retention time of replicates of standard preparation should not be more than 1.0 %

2.     % RSD for area of replicates of standard preparation should not be more than 2.0 %

3.     Theoretical plates for all standards injections should not be less than 2000. Report Theoretical plates of first replicate of standard preparation.

4.     Tailing factor for all standard injections should not be less than 2.0. Report tailing factor of first replicate of standard preparation.

 

 

Calculate Content of Febuxostat (mg/Tablet) for both sample preparations independently by using formula described below:

 

 

(SPL area)                                       W1   10   100  100    P

   ………………………………….     x ……x… x….. x…  x…. x w3 (gm)

(Average area of 5 STD replicates)   100 100  W2   10    100

 

For % Assay = (mg/per Tablet) (100)/(Label Claim i.e. 40) Where,

W1  = Weight of Febuxostat standard taken in mg for standard prep.

W2 = Weight of sample taken in gm for Sample preparation

W3  = Average weight of Tablets in grams

P     = % Purity of Hydrochlorothiazide on as such basis. Acceptance Criteria

Assay of Febuxostat in should be between 36 to 44mg per Tablet.

% Assay of Febuxostat should be between 90.0 to 110.0

 

METHOD DEVELOPMENT DETAILS^6,7

In order to optimize the LC conditions for the estimation of Febuxostat in tablet the following trials were performed. Initially a mobile phase consisting of 10mM Potassium Di-hydrogen Orthophosphate (pH 3.5): Methanol (60:40%v/v) at a flow rate of 1.0mL/min was used on an Agilent Eclipse C18 column (250 x 4.6 ) 5 micron column at ambient temperature using mobile phase as diluent, Febuxostat did not elute under these conditions.

 

Trial 1

 

In the next trial, same column was employed but the mobile phase was changed to mobile phase consisting of 20mM Potassium Di-hydrogen Orthophosphate (pH 3.5): Acetonitrile (60:40% v/v) at a flow rate of 1.0mL/min was used on an Agilent Eclipse C18 column (250 x 4.6 ) 5 micron column at ambient temperature.

 

Trial 2

 

Febuxostat eluted with at 9.104 with theoretical plates 11273 (limit NLT 2000 and Tailing factor 1.05 (NMT)

 

2) Which well within the limit but as retention time is more and will time consuming during analysis therefore focusing on reduction of retention time drastically. So to reduce the time for elution as well as cost of analysis.

 

Following changes are done:

1)    Methanol solvent is introduced in some portion of acetonitrile.

2)    Buffer: ACN: methanol. (40:40:20).

3)    Concentration of salt increased from 20 to 30Mm

4)    pH of buffer is shifted from 3.5 to 3.0

5)    Column length is decreased. From 250 to 150 cm

 

Lambda max is confirmed by ultra violet spectroscopy. spectra is observed as below.

 

Lambda max is determined before conducting next trial and found 317.4nm i.e. equivalent to 317nm As designed above trial conducted with mobile phase consisting of 30 mM Potassium Di-hydrogen Orthophosphate (pH 3.0): Acetonitrile: Methanol (50:40:10% v/v) at a flow rate of 1.0mL/min was used on an Agilent XDB C18 column (150 x 4.6) column at 40°C.

 

Febuxostat eluted with at 4.498 with theoretical plates 8805 (limit NLT 2000) and Tailing factor 1.12 (NMT 2) which well within the limit. Thus, further injection of same std is done and to conclude precision. Corresponding area, RT and system suitability parameters observed.

 

Method Validation:^8,9

A)   SPECIFICITY AND SYSTEM SUITABILITY:

Specificity demonstrated by observing interference of mobile phase (Diluent). System suitability parameters (% RSD of area, Retention time, Theoretical Plates and Tailing factor) demonstrated by injecting standard preparation in replicate.

 

Table No.1: Specificity and System Suitability

 

No interference observed from Mobile phase (Diluent) Filter paper used for analysis found comfortable as per limit % RSD.

 

Conclusion – Method found specific and capable to achieve System suitability.

 

B)   PRECISION:^10,11

Repeatability: The repeatability was demonstrated by preparing the standard solution at 40ppm concentration and six independent consecutive sample preparations at 40ppm. System suitability found within limit. Relative standard deviation of assay value for six preparations found within 2%

 

 

Table 2: Repeatability

 

System Suitability:

Average std. area = 56269.3638

 

Conclusion of repeatability:

%RSD for repeatability found 0.79 which well within limit therefore method is repeatable.

 

C)   Intermediate Precision:^12,13

The Intermediate Precision was demonstrated by preparing the standard solution at 40 ppm concentration and six independent consecutive sample preparations at 40 ppm by other person on other day with other set of chemicals. System suitability found within limit. Relative standard deviation of assay value for six preparations found within 2%.

 

Percentage variation of average assay values obtained via repeatability and intermediate precision found within limit.

 

 

Table 3: Intermediate Precision

 

System Suitability:

Average std. area = 53617.4402

 

Conclusion of Intermediate precision:^14,15,16

Percentage RSD for 6 replicate independent analyses by changing various objects found 0.52 % which well within limit therefore method is intermediately reputable.

 

Percentage RSD for 12 observations (6 of repeatability and 6 of intermediate precision) found 0.93 %which   well within limit therefore Based on both experiment Method found Precise

 

ACCURACY:

To determine the accuracy of the method, recovery studies were carried out in triplicate by using different concentrations of pure drug in the pre analyzed samples with 3 different concentrations of sample that consists of 80 %, 100 % and 120 % of the pure drug. The accuracy was expressed as the percentage analytes recovered.

 

Accuracy sample_80%

 

 

Accuracy sample_100%

 

 

Accuracy sample_120%

 

 

Table 4:   Accuracy

 

 

Conclusion – All parameter found within Limit therefore Method found Accurate.

 

LINEARITY AND RANGE:^17,18,19

From the standard stock solution, the various dilutions of Febuxostat in the concentration of 160.0, 200.0, 240.0 ppm three level standard solutions of each were prepared. The solutions were injected using 20μL injection volumes in to the chromatographic system at the flow rate of 1.0ml/min and the effluents were monitored at 317nm, chromatograms were recorded. Calibration curve of Febuxostat was obtained by plotting the peak area ratio versus the applied concentrations of Febuxostat by using average of each sample. The linear correlation coefficient (R2) was found to be 1.000 & %y intercept is -0.0035 %

 

 

Linearity Std 80

 

 

Linearity std 120%

 

 

Linearity Std 100%

 

Correlation-1.000

% Y intercept= -0.0035

 

Table No. 5 Linearity and range

 

Conclusion – Method found Linear within the range 80 % to 120 % of working level

 

A) LIMIT OF DETECTION AND LIMIT OF QUANTITATION (LOD AND LOQ)^20,21,22

The limit of detection and limit of quantification means the lowest concentration of analytes in the sample are detected and quantified. LOD and LOQ was found as listed below

 

Table 6: Limit of Detection and Limit of Quantitation

 

B) ROBUSTNESS:^23,24,25

Robustness of the method was determined by intentionally changing some operating conditions such as flow rate and wavelength. The flow rate as per the developed method is 1.0ml/min. It has been purposely changed to 0.8ml/min and 1.2ml/min and the chromatogram was developed as well as the wavelength of developed method is 317nm. It has been purposely changed to 315nm and 317nm and the chromatogram was developed.

 

SPL 0.8ml/min

 

 

SPL Wavelength =317nm

 

SPL 1.2ml/min

 

System Suitability^26,27,28

 

Table 7: Robustness Flow rate = 0.8 ml /min

 

System Suitability

 

Table 8: Robustness: Detection wavelength = 317 nm

 

% Cumulative RSD of % assay observed for changing parameters calculated and found within limit i.e. below 3%

 

Conclusion – Method found Robust^29,30

 

Non-conformance:

Specify the non-conformance (if any) observed during method validation.

 

CONCLUSION:

This isocratic RP-HPLC analytical method used for determination of assay of Febuxostat in Tablet is within acceptance criteria for the analytical parameters such as Specificity and System suitability, Linearity and Range, Precision, Accuracy and Robustness. Hence method stands validated.

 

ACKNOWLEDGEMENT:

I would like to Acknowledge and give my gratitude to Instavision Laboratory and Services Satara for providing me with the opportunity to finish all of this laboratory work. Their assistance and director Dr. Ajit B. Ekal and Miss. Sonali. T. Dhumal helped me get through all of the stages of my project. Ajit B. Ekal is also to be thanked for his inspirational guidance, affectionate encouragement, and never-failing enthusiasm, without which this research would not have seen the light of day. Prof. V. K. Redasani (Principal YSPM, YTC, Faculty of Pharmacy, Satara) for his constructive comments, rigorous assistance, and persistent motivation would not suffice to express my heartfelt gratitude. Above all, I'd like to express my gratitude to my family for their inexhaustible generosity and clementines and grass up on me, for being my continuous companion, the most powerful source of motivation and inspiration and my ultimate guard.

 

REFERENCES:

1.      Avinash M. Bhagwat, Anand P. Khadke, Anuradha M. Patil, Snchal V. Tarade. Quality preparation work: the basic need of analysis. Safe handling of laboratory. European Journal of Biomedical and Pharmaceutical Science. 2017; 4(4): 158-165

2.      Chatwal GA. Instrumental Methods of Chemical Analysis, Himalaya Publishing House, Firstedition, 2.167-2.171

3.      Horiuchi H, Ota M and Kobayashi M. A comparative study on the hypouricemic activity and potency in renal xanthine calculus formation of two xanthine oxidase/xanthine dehydrogenase inhibitors: TEI-6720 and allopurinol in rats. Res Commun Mol Pathol Pharmacol. 1999; 104:307-319.

4.      ICH, Q2A validation of analytical procedure: Methodology International Conference on Harmonization, Geneva, October 1994.Bisht M and Bist SS. Febuxostat: a novel agent for management of hyperuricemia in gout. Ind J Pharm Sci. 2011; 73(6): 597-600

5.      Ch, Shankar, R Suthakaran, and Prem Kumar B. A New RP-HPLC Method Development and Validation for the Simultaneous Estimation of Febuxostat And Ketorolac in Bulk and Tablet Dosage Form. International Journal of Biomedical Investigation. 2018; 1(2): 1-9. https://doi.org/10.31531/2581-4745.1000113

6.      ICH Harmonised Tripartite Guideline, Q2 (R1), Validation of Analytical Procedures: Text and Methodology. International Conference on Harmonisation, Geneva. 2005; 1-13.

7.      Kushwah D. Study of impurity carryover and impurity profile in Febuxostat drug substance by LCMS/MS technique. J Pharm Biomed Anal. 2011; 50(6):749-757.

8.      Nageswara Rao K, Ganapaty S and Lakshmana Rao A. Development and validation of RP-HPLC method for estimation of Febuxostat in bulk and tablet dosage form, International Journal of Research in Pharmacy and Chemistry, 2(4), 2012, 1104 1108"*

9.      Zhang Cong, Wang Shao-jie, Rong-li MA, Meng Ping, Zhang Tian-hong.  Determination of content of Febuxostat and its related substances by HPLC. 2010.

10.   Becker M.A., Kisicki J, Khosravan R, WuJ, Mulford D, Hunt B, Mac Donald P, JosephRidge N., Febuxostat (TMX-67), a novel, nonpurine, selective inhibitor of xanthine oxidase, is safe and decreases serum urate in healthy volunteers, Nucleosides Nucleotides Nucleic.

11.   Mustakhusen H. Kadivar, Praveen Kumar Sinha, Dharmendra Kushwah, Pijush Jana, Hitesh Sharma, Atul Bapodra, Study of impurity carryover and impurity profile in Febuxost at drug substance by LC–MS/MS technique, Journal of Pharmaceutical and Biomedical Analysis. 2011; 56(4): 749-757

12.   Cooper N, Khosravan R, Erdmann C, Fiene J, Lee JW. Quantification of uric acid, xanthine and hypoxanthine in human serum by HPLC for Pharmacodynamic studies, J Chromatogr B Analyt Technol Biomed Life Sci. 2006; 837: 1-10.

13.   Khosravan R, Wu JT, Joseph-Ridge N, Vernillet L. Pharmacokinetic Interactions of Concomitant Administration of Febuxostat and NSAIDs. J Clin Pharmacol. 2006; 46: 855-866

14.   http://en.wikipedia.org/wiki/Brexpiprazole

15.   Paramdeep B., Mohd Salman, H., Siddiqui H., Abdul Ansari M., Tariq Mehmood and Kuldeep Singh, A Simple UV Spectrophotometric method for the determination of Febuxostat in bulk and pharmaceutical formulations. IJPSR. 2011; 2(10): 2655- 2659.

16.   Michael E Swartz, Brian J Murphy. Ultra performance liquid chromatography, Tomorrow’s HPLC technology today Laby, Plus International. 2004; 18: 6-9

17.   Aimee N Herrmann, Richard A Henry, importance of controlling mobile phase pH in reversed phase (RP), Technical bulletin 99-06.

18.   Burd RS and Lobias JD. Ketorolac for pain management after abdominal surgical procedures in infants. South Med J. 2002; 95: 331- 333.18.18.

19.   International Conference on Harmonization (2005) ICH Harmonized Tripartite Guidelines (Nov 2005), Validation of Analytical Procedures, Text and Methodology Q2 (R1). ICH, Geneva.

20.   Ranjith, K, M V Basaveswara Rao, D Rama Sekhara Reddy, and T E G K Murthy. Development and Validation of Related Substances Method for Febuxostat Tablets by RP-HPLC. Indian Journal of Research in Pharmacy and Biotechnology. 2017; 5(3): 229-34.

21.   Chavan Pooja Ajit, Avinash Mahadeo Bhagwat, Atul Prabhakar Chaudhari. An important concept of Quality Assurance in Pharmaceutical Industry; Asian Journal of Research in Chemistry.

22.   Chavan Pooja Ajit, Avinash Mahadeo Bhagwat, Atul Prabhakar Chaudhari. CAPA: An important concept of Quality Assurance in Pharmaceutical Industry. Asian Journal of Research in Chemistry. 2021; 14(5): 357-2.

23.   Chavan Pooja Ajit, Shelar Reshma Dattatraya, Shelake Pallavi Ramchandra, Avinash Mahadeo Bhagwat, Ajit Bhiva Ekal. RP-HPLC Method Development and Validation of Tadalafil in Tablet Dosage form. Asian Journal of Research in Chemistry. 2021; 14(5): 380-8.

24.   Abhijeet Shedmake, Priyanka Jadhav, Suvidha Mane, Vaibhavi Rao, Avinash Bhagwat, Vivekkumar Redasani. Determination, Method Development and Validation of Six Nitrosamine Impurities in Purified water by using LC-MS/MS. Asian Journal of Research in Chemistry. 2023; 16(5): 377-2.

25.   P. Balan, I. Carolin Nimila, M. Lakshmi Prasanna, M. Vanaja Rani, S. Rajasekar. RP-HPLC Method Development and Validation of Etodolac and Paracetamol in Combined Dosage Form. Asian J. Research Chem. 2011; 4(7): 1073-1076

26.   Prabhu S, Vijay Amirtharaj R, Senthilkumar N. Simultaneous RP-HPLC Method Development and Validation of Cefixime and Ofloxacin in Tablet Dosage Form. Asian J. Research Chem. 2010; 3(2): 367-369.

27.   M.M. Eswarudu, T. Narendra Chary, Sunil Junapudi, M. Sushma. RP-HPLC Method Development and Validation for Simultaneous Estimation of Irbesartan and Hydrochlorothiazide in Pharmaceutical Dosage Form. Asian J. Research Chem. 2012; 5(4): 472-476.

28.   Vatchavai Bhaskara Raju, Bonthu Mohan Gandhi, Kamatham Srinivas Sumanth, Kolli Srinivas, Tupakula N Venkata Lakshmi Neeraja. RP-HPLC Method Development and Validation for Simultaneous Estimation of Telmisartan and Ramipril in pure and Pharmaceutical Dosage forms. Asian J. Research Chem. 2017; 10(2):179-185.

29.   K. Vijaya Sri, S. Ravinderreddy, K. Suresh. Rapid RP-HPLC Method Development and Validation for Analysis of Raltegravir in Bulk and Pharmaceutical Dosage Form. Asian J. Research Chem. 2015; 8(5): 335-339

30.   Manoharreddy Epuru, Ravinder Reddy V, Nagarjuna Reddy P, Kishore Ravada, Jajula Kashanna, V V N Kishore Pilli. RP-HPLC Method Development and Validation for Simultaneous Estimation of Escitalopram Oxalate and Atazanavir Sulphate in Bulk and Dosage Form. Asian Journal of Research in Chemistry. 2023; 16(2): 163-8

31.   Chavan Pooja Ajit, Shelar Reshma Dattatraya, Shelake Pallavi Ramchandra, Avinash Mahadeo Bhagwat, Ajit Bhiva Ekal. RP-HPLC Method Development and Validation of Tadalafil in Tablet Dosage form. Asian Journal of Research in Chemistry. 2021; 14(5): 380

 

 

 

 

Received on 11.01.2024                    Modified on 15.04.2024

Accepted on 28.05.2024                   ©AJRC All right reserved