Reverse Phase High Performance Liquid Chromatographic Method for the Analysis of Etoricoxib in Pharmaceutical Dosage Form

 

Indrani Bhattacharyya1*, Subhra Prakash Bhattacharyya1, Subrata Sen2 and Tapas Ku Laha2

1Department of Pharmaceutical Chemistry, Dr B.C Roy College of Pharmacy and Allied Health Sciences, Meghnath Shah Sarani, Bidhan Nagar, Durgapur-6, West Bengal, India

2College of Pharmaceutical Sciences, Mohuda.

*Corresponding Author E-mail: indrani.das@rediffmail.com

 

ABSTRACT

A rapid and sensitive reverse phase HPLC method is depicted for the qualitative and quantitative assay of Etoricoxib in pharmaceutical dosage forms. Etoricoxib was chromatographed on a reverse phase C18 column with a mobile phase consisting of methanol: phosphate buffer (pH-7.8) in the ratio of 90:10 v/v.The mobile phase was pumped at a flow rate of 1ml/min. Mirtazapine was used as an internal standard and the eluents were monitored at 235 nm. The retention time of the drug was 3.428 min.With this method, linearity is observed between area under curve (AUC, expressed in mV.min) and concentration of Etoricoxib in the injected solution, in the range of 10-200 g/ml.The method was found to be applicable for analysis of drug in tablets. The results of the analysis were validated statistically.

 

KEYWORDS: Etoricoxib, Reverse phase HPLC, Tablets.

 


INTRODUCTION:

Etoricoxib1,2 is a new NSAID, which is chemically 5-chloro-6-methyle-3-[4-(methylesulfonyl) phenyl]-2, 3-bipyridine.It is active at low dose and has less gastric toxicity3. It exhibits synthesis of prostaglandins by inhibiting the activity of the enzyme; cycloxygenase-2(cox-2)4.It has highest cox-2 selectivity and better safety profile. Prostaglandins are involved in mediating inflammation, swelling, pain and fever.Etoricoxib is preferred over conventional NSAID as they may lead to serious gastrointestinal complications such as ulcer, severe bleeding and perforation, resulting in hospitalization and even death5. It is mainly used for the osteoartherities, rheumatoid artherities and acute gouty artherities6-8.The drug is available in tablet form (60mg, 90mg and 120mg) and is not official in any pharmacopoeia. A few methods of analysis of Etoricoxib have been reported using different techniques such as validated liquid chromatographic ultraviolet method for the quantization of Etoricoxib in human plasma using liquid-liquid extraction9, solid-phase extraction-liquid chromatography tandem mass spectrometry10-12, spectrophotometric estimation of Etoricoxib in bulk drug and dosages forms13. Most of these methods are considered tedious. The HPLC methods using the most commonly available columns and detector like UV are preferred.

 

The present study describes the determination of Etoricoxib in pharmaceutical dosage forms by using RP-C18 column with UV detectors. Owing to the widespread use of HPLC in routine analysis, it is important that well validated HPLC method for the estimation of Etoricoxib in different pharmaceutical dosage forms.

 

MATERIAL AND METHOD:

The pure Etoricoxib we used for the development of analytical method was gifted by Torrent pharmaceutical Ltd Gujarat India. Methanol and water were of HPLC grade (Merck).All other regents were of AR grade. An isocratic HPLC (Waters India, USA) with a single Waters 510 pump, Waters 486 tunable absorbance detector and RP-C18 column (Bondapak C18 2504.6mm, packed with 5m particle size) was used. The HPLC system was equipped with Millennium32software.

 

Chromatographic conditions

The composition of the mobile phase is methanol and phosphate buffer at ph 7.8 in the ratio of 90:10 v/v. The mobile phase was filtered before use through a 0.4m membrane filter and degassed for 30 min.

 

The components of the mobile phase were pumped from the solvent reservoir to the column at a flow rate of 1ml/min that produced column back pressure 140-150 kg/cm2.Ambient column temp was maintained. The eluents were monitored at 235nm.

 

Table-1 Calibration of the proposed method

SL.

NO

CONC (μg/ml)

Mean Peak Area Ratio (n = 6)

% R. S. D

1.

10

0.389542

0.06

2.

20

0.810043

0.23

3.

30

1.129712

0.96

4.

40

1.518280

0.05

5.

50

1.909756

0.71

6.

60

2.125662

0.64

7.

70

2.602046

0.79

8.

80

3.093361

0.19

9.

90

3.389160

0.78

10.

100

3.728239

1.01

11.

150

5.647506

0.88

12.

200

7.624929

0.91

 

 

Table-2 Precision of the proposed method

 

CONC OF MIRTAZAPINE (μg/ml)

OBSERVED CONC OF MIRTAZAPINE (μg/ml)

INTRA DAY (MEAN) n=6

%

R. S. D

INTER DAY (MEAN) n=6

%

R. S. D

50

50.40

0.56

50.32

0.81

60

58.08

0.98

58.02

1.41

70

68.99

0.25

68.98

0.23

 

 

 

 

 

 

 

 

 

 

Drug and internal standard solution:

A pure sample of Etoricoxib procured from Torrent pharmaceutical ltd, Gujrat was used as reference standard in the study. About 50mg of Etoricoxib was weighed accurately and transferred into a 50ml volumetric flask and dissolved in 25ml of the mobile phase. Then the volume was made up with a further quantity of mobile phase to get 1mg/ml solution. Following this the solution was sonicated for 30min to degas it.Subsiquent dilution of this solution ranging from 10-200g/ml were made in 10ml volumetric flask after addition of 1ml Mirtazapine solution (100g/ml) as an internal standard to each dilution.20l of the solution was injected each time into the stream of mobile system at a flow rate of 1ml/min.each of the dilution was injected six times into the column and the corresponding chromatograms were obtained. From these chromatograms, the area under the peaks of the drug and internal standard were noted .Using these values, the mean ratio of peak area of the drug to that of the internal standard for each dilution was calculated. The regression of the drug concentration was computed. This regression equation was used to estimate the amount of Etoricoxib in the pharmaceutical dosage forms.

 

Solutions containing 50-70ug/ml of Etoricoxib were subjected to the proposed HPLC analysis to check the intra-day and inter-day variation of the method. The recovery studies were carried out by adding known amounts of Etoricoxib to the preanalyzed sample and then analyzing them by the proposed HPLC method.

 

Estimation of Etoricoxib in the Tablet dosage form:

Two commercial brands of tablets (Etoxib of Unichem and Hicox of Systopic) were chosen for testing suitability of the proposed method to estimate Etoricoxib in tablet formulation. Twenty tablets were weighed and powdered. An accurately weighed portion of this powder equivalent to 50mg of Etoricoxib was transferred to a 50ml volumetric flask containing 25ml of mobile phase. The content of the flask were allowed to stand for

 

6 hours with intermittent sonication to ensure complete suitability of the drug and then filtered through 0.4 membrane filter. From the filtrate different aliquots were taken in separate 10ml volumetric flask. These solution were spiked with suitable volume of internal standard solution, such that the concentration of each solution was 100ug/ml.The contents of the flask was made up to the volume with the mobile phase and mixed well. Each of this solution (20L) was then injected 6 times into the column. The mean peak area ratio of the drug to the internal standard of 6 such determinations was calculated and the drug content in tablets was quantified using the regression equation obtained for the pure sample.

 

RESULT AND DISCUSSION:

To achieve precise component peaks with good resolution under isocratic conditions mixtures of ethanol and phosphate buffer in different combination were tested as mobile phase on a C18 stationary phase. A binary mixture of methanol and phosphate buffer (pH7.8) in 90:10v/v proportion was proved to be the most suitable of all combination since the chromatographic peaks were better defined and resolved and almost tailing with this system. Though the structure of Mirtazapine is not to similar to Etoricoxib,it was chosen as an internal standard, because it showed better peak shape and peak location compared to other potential internal standard. Under the above mentioned chromatographic condition the retention time obtained for Etoricoxib and internal standard were 3.428 and 5.240 min respectively. A model chromatogram was shown in the figure below.

 

Each of the samples was injected six times and same retention times were observed in all cases. The ratio of peak area of Etoricoxib to peak area of internal standard for different concentration set up as above were calculated and the average values for six such determination are shown in Table-1.

 

Figure1

 

 


Table-3 Recovery Data of Etoricoxib

 

Amount of drug added(g)to solution of pure drug /tablet

formation

Recovery from drug solution

Recovery from tablet formulation

Mean(S.D) Amount(g) Found (n=6)

Mean(S.D) %recovery(n=6)

Mean(S.D) Amount (g) Found (n=6)

Mean(S.D) %recovery(n=6)

16

15.880.61

99.250.32

15.820.73

98.550.78

20

29.950.54

99.760.63

19.850.11

99.380.45

24

23.840.71

99.24.051

23.680.81

98.520.16

 

 

 

 

 

 

 

 

 

Table-4 Assay of Etoricoxib dosage form

Brand name of the tablet

Labeled amount of drug (mg)

Mean(S.D)amount (mg) found by the proposed method

% Mean( S.D) of labeled amount (n=6)

Etoxib

60

59.870.08

99.770.81

Hicox

60

59.210.22

98.690.49

 


The peak area of both the drug and internal standard were reproducible as indicated by low coefficient of variance (1.01%). A good linear relationship (r=0.9995) was observed between the concentration of Etoricoxib and the respective ratio of peak areas.

 

The drug content in the tablets was quantified using the proposed analytical method. The mean amount of Etoricoxib in two different brands of tablet dosage form is shown in Table-4.The absence of additional peaks in the chromatogram indicates non-interference of the common exipients used in the tablets. It can be concluded that the proposed HPLC method is sufficiently sensitive and reproducible for the analysis of Etoricoxib in pharmaceutical dosage forms within a short analysis time. The method was duly validated by the required parameters.

 

CONCLUSION:

Development of new analytical methods for the determination of drugs in pharmaceutical dosage forms is more important in pharmacokinetic, toxicological and biological studies. The aim of the present study was to develop simple, fast and sensitive HPLC method for the determinations of Etoricoxib.

 

The method described herein is simple validated assay procedures that can readily be used in any laboratory for the quantitative determination of Etoricoxib. Analytical figures of merit demonstrated during the method validation protocol compare well with those of known methods for the determination of Etoricoxib. The assay procedure was simple with satisfactory precision (less than 3.5) and accuracy in terms of relative error (less than 2.0). We believe that this method fulfill experimental and clinical requirements for determining the drugs and can be applied for any study. Concentration of buffers used in this method was very low and pH used also reliable and doesnt produce any troubleshoot to column and instrument. Practically, low cost of single analysis are central features of routine laboratory tests, and the herein described RP-HPLC method, because it uses low cost solvents and is easily affordable by clinical laboratories equipped with standard high-performance liquid chromatography systems. So the developed method was very useful in the determination of Etoricoxib.

ACKNOWLEDGEMENT:

The authors are grateful to Torrent pharmaceutical Ltd, Gujrat, for providing gift sample of pure Etoricoxib. Above all, the authors would like to offer their gratitude to the authorities of Dr B.C Roy college of pharmacy and allied providing all facilities.

 

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Received on 30.04.2009 Modified on 25.07.2009

Accepted on 04.08.2009 AJRC All right reserved

Asian J. Research Chem. 2(3): July-Sept., 2009, page 297-299