DEVELOPMENT AND APPLICATION OF DIFFERENCE SPECTROPHOTOMETRIC METHOD FOR THE DETERMINATION OF FEBUXOSTAT IN TABLETS

DEVELOPMENT AND APPLICATION OF DIFFERENCE SPECTROPHOTOMETRIC METHOD FOR THE DETERMINATION OF FEBUXOSTAT IN TABLETS

M. Sheth*, S. Joshi and M. Patel

Pharmaceutical Analysis and Quality Assurance Department, K.B. Raval College of Pharmacy, Shertha, AT-Post. Kasturinagar, B/h. Iffco Township, Dist.-Gandhinagar-382 423, Gujarat, India

ABSTRACTA simple, rapid, sensitive and cost effective difference spectrophotometric method have been developed for the determination of Febuxostat in its tablet dosage form. The method is based on the induced spectral changes upon changing the pH of the medium that differ in their UV spectra. Difference spectrum, obtained by keeping Febuxostat in 0.1N NaOH in reference cell and same  in 0.1N HCl in sample cell, showed two characteristic peaks at 260nm and 315nm with positive and negative absorbance respectively and viceversa. Difference of absorbance between these two maxima was calculated to find out the amplitude, which was plotted against concentration. The calibration curve is linear over the concentration range of 5-25 µg/ml (r2 = 0.999), with a detection limit of 0.58µg/ml. The method was successfully applied to the commercial pharmaceutical drug without interference from common ingredient accompanying the drug along with its dissolution profile and very cost effective as its prevents the use of hazardous and costly organic solvents

Amplitude

INTRODUCTION:Febuxostat,  antigout  agent  is  chemically 2- [3- cyano-4-  (2- methlypropoxy) phenyl]-  4- methlythiazole-  5  - carboxylic  acid (fig. 1). It  is  a  non  purine selective  inhibitor  of xanthine oxidase ¹. It  inhibits  both  oxidized  and  reduced forms  of  xanthine oxidase ^{2, 3} and  has very  less  effects  on  other  enzymes  of purine and pyrimidine metabolism ^{3, 4}. Based  on  the  literature  survey  it  shows that very few analytical methods have been reported for the estimation of Febuxostat which includes UV (Drug dissolution ⁵ and determination ⁶), HPLC ⁷,  GC ⁸  and  LC/MS/MS (Impurity profiling) ⁹.

The aim of the study is to develop a simple, sensitive, accurate,  precise and cost effective method  for  determination  of  febuxostat in  pharmaceutical  formulations  and  bulk drugs  using  UV  spectro- photometer without  using  costly  and  hazardous organic  solvents.

FIG. 1: STRUCTURE OF FEBUXOSTAT

Difference  spectrophotometric  assay  measure  the  difference  absorbance  between  two  equimolar  solutions  of  the analyte  in  different  chemical  forms, which  exhibit  difference  spectral characteristics.  The  simplest  and  most commonly  employed  technique  for altering  the  spectral  properties  of analyte  is  the  adjustment  of  the  pH  by means  of  aqueous  solution  of  acids  and  alkali.

MATERIALS AND METHOD: A   Simadzu  UV  Spectro-photometer  1800  with  1.0  cm  matched  quartz  cells was  used.  Febuxostat  bulk  drug  was obtained  from  Cadila  Healthcare  Ltd., Ahmedabad.  Tablets (Urifix 40mg)  were obtained  from  the  market  (manufactured by Precise Biopharma Ltd.)  Sodium hydroxide  and  Hydrochloric  acid  (were from Merck, Mumbai, India) (0.1M Solution),  Water  was  always  distilled. The  year  of  experimentation  was  2012, at K.B.Raval College of Pharmacy, Shertha, Dist. -Gandhinagar-382423, Gujarat,  India.

RESULT AND DISCUSSION:

Calibration: Stock  Febuxostat  solution was  prepared  by  dissolving  10 mg  of working  standard  in  10 ml  of methanol. Working  standard  solutions  with concentration  ranging  from  5-25  µg/ml were  prepared  by  transferring appropriate  volume  of  stock  solution  to 10ml  volumetric  flask  in  duplicate.  The volume  was  then  adjusted  with  0.1M HCl  and  0.1M  NaOH  to  give  a series of  equimolar  solutions  of  Febuxostat. Difference  spectra were  obtained  by keeping  acidic  form  (in 0.1M HCl)  in reference  cell  and  basic  form  (in 0.1M NaOH)  in  sample  cell  and  vice  versa. Difference  of  absorbance  between 260nm  and  315nm  was  calculated  to find  out  the  amplitude (table 1)

TABLE 1: DIFFERENCE STANDARD CURVE OF FEBUXOSTAT IN 0.1M HCl WITH RESPECT TO 0.1M NaOH

TABLE 2. DIFFERENCE STANDARD CURVE OF FEBUXOSTAT IN 0.1M NaOH WITH RESPECT TO 0.1M HCl

FIG. 2: OVERLAY SPECTRA OF FEBUXOSTAT IN 0.1M HCl WITH RESPECT TO 0.1M NaOH

FIG. 3. CALIBRATION CURVE OF FEBUXOSTAT IN 0.1M HCl W.R.T. 0.1M NaOH

FIG. 4. OVERLAY SPECTRA OF FEBUXOSTAT IN 0.1M NaOH WITH RESPECT TO 0.1M HCl

FIG. 5. CALIBRATION CURVE OF FEBUXOSTAT IN 0.1M NaOH W.R.T. 0.1M HCl

Procedure for the assay of Febuxostat from tablet: The  average  mass  of  10 tablets  was  determined  and  was ground in a  mortar.  An amount  of  powder (accurately weighed)  equivalent  to  10mg Febuxostat  was  transferred  in  10ml volumetric  flask  and  made  up  to  the mark  with  methanol.  The content  of  the flask  was  sonicated  for  10min  and  then the  solution  was  filtered  through Whatmann  filter  paper.

Then  1ml  of  the  filtrate  was  transferred  to  10ml volumetric  flask  in  Methanol.  From the above  solution  1 ml  of  solutions  were transferred  to  10 ml  volumetric flask  in duplicate  and  made  up  to  mark  with 0.1M NaOH  and  0.1M HCl  respectively. The  absorbance  difference  between  the 0.1M HCl  solution  and  equimolar  0.1M NaOH solution  was  measured  at  206nm and  315nm  by  placing  basic  solution  as reference  and  acidic  solution  as  sample. The  content  of  the  tablet  is  calculated from  the  calibration  curve  or  using  the corresponding  regression  equation  in (table 3)

TABLE 3: ASSAY OF FEBUXOSTAT FROM MARKET FORMULATION

Validation Parameters: Estimation  of  Febuxostat  was  validated as  per  ICH  guidelines ¹⁰ .

Accuracy:  To  check  the  accuracy  of the  developed  methods  and  to  study  the  interference  of  formulation  additives,  analytical  recovery experiments  was  carried  out  by  standard  addition  method.  Total  amount of  drug  found  and  percentage  recovery was  calculated  by  adding  0%,  80%, 100%  and 120%  standard  and    results were  reported  in  table 4.

TABLE 4. RECOVERY STUDY OF FEBUXOSTAT

Precision (inter-day and intra-day precision):  precision  of  the  method  was  checked  by  assay  the  sample solution  on  sameday  at an  interval  of one  hour(intraday precision)  for  three hours  and  on  three  different  days (interday precision)  the  result  was reported  in  table 5. This  study indicates  that  the  solutions  can  be analyzed  within  48-72 hr  without  having any  adverse  effect  on  chemical  stability of  the  drug  in  acidic  and  basic medium.

TABLE 5. INTERDAY AND INTRADAY PRECISION STUDY OF FEBUXOSTAT

Limit of detection (LOD) and limit of quantitation (LOQ): The  LOD  and  LOQ  were  separately determined  based  on  the  standard deviation  of  response  of  the  calibration curve.  The  standard  deviation  of  Y-intercept  and  slope  of  the  calibration curves  were  used  to  calculate  the  LOD and  LOQ  by  using  the  equations  3.3σ/s for  LOD  and  10σ/s  for  LOQ,  where  σ stands  for  standard  deviation  of  Y-intercept  and  S   stands  for  slope  of  the calibration  curve.  The  results  of  the same  were  given  in  table 5.

Application of developed difference spectroscopic method for dissolution study of Febuxostat: Dissolution  is  an  official  test  used   by pharmacopoeias  for  drug  evaluation release  of  solid  and   semisolid  dosage forms,  and  it   is  routinely  used  in Quality  Control  (QC)  and  Research  & Development  (R&D).

The  purpose  of   in  vitro  dissolution  studies  in  QC  is batch  to  batch  consistency  and  detection  of  manufacturing  deviation while   in  R&D  the  focus  is  to   provide some  predictive  estimate  of  the  drug release  in  respect  to  the  in  vivo performance  of  a  drug product.

For  QC, an  over-discriminatory  test  might  be suitable  to  detect  even  small  production deviations.  However,  for  prediction  of the   in vivo  performance  of  drug  product  a  dissolution  test  should be sensitive  and  reliable ¹¹. A  review  of the  literature  found  a  simple  UV spectrophotometric  method  reported  for the  determination  of  Febuxostat  in  raw materials  and   in  vitro  drug  dissolution studies ⁵, but,  it  is  costly  as  it  require methanol  as  the  solvent.  So,  it  is  better to  use  another  cost  effective  method  for  the  same  purpose.  The above developed  difference  spectroscopic method  is  suitable  for  the  dissolution purpose.  Febuxostat  from  tablets containing  40 mg  of  this API,  using Phosphate  buffer  pH  6.8  with  0.0675% SLS  as  the  dissolution  medium.  RPM was  set  at  75  and  the  temperature  was maintained  at  37±0.5°C.  Final  dilution of  the  samples  were  done  in  0.1M NaOH  and  0.1M HCl  and %  cumulative Release  of  the  drug  was  determined.

FIG. 6. DISSOLUTION PROFILE OF FEBUXOSTAT

ACKNOWLEDGEMENT: The  authors  are  thankful  to  Cadila Healthcare  Ltd,  Ahmedabad   for  providing  the  free  gift  samples  of Febuxostat  for  the  research  work.

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