Int J Pharm Pharm Sci, Vol 6, Issue 7, 470-474Original Article

SIMULTANEOUS ESTIMATION OF FEBUXOSTAT AND NAPROXEN IN SYNTHETIC MIXTURE BY REVERSE PHASE HIGH PERFORMANCE LIQUID CHROMATOGRAPHY METHOD

MS. KRUPA THULA*, PATEL RESHMA A.*, DILIP G. MAHESHWARI

(Head of the Department) L. J. Institute of Pharmacy, S.G. Road, Ahmedabad-382210
Email: krupathula@yahoo.com, patelreshma22@gmail.com

Received: 09 May 2014 Revised and Accepted: 19 June 2014

ABSTRACT

Objective: To develop simple, precise, rapid, and accurate RP-HPLC method for the simultaneous estimation of Febuxostat and Naproxen in synthetic mixture. To perform complete validation of newly developed analytical methods as per ICH(Q2) R1 [32] Guideline.

Methods: In RP-HPLC method for Febuxostat and Naproxen, chromatographic separation was carried out on Shimandzu LC-20AT, Hypersil BDS column (250mm x 4.6mm, 5 μm) using mobile phase ACN: Methanol: Phosphate Buffer (50:35:15 v/v) pH 5, detected at 288 nm, with flow rate 1.0ml/min and injection volume 20 μL.

Results: For RP- HPLC Linearity of Febuxostat and Naproxen were found to be 4 – 12 μg/ml and 25 - 75 μg/ml respectively. For both the developed and validated methods the %RSD was found to be less than 2% and the % recovery was found to between 98-102 %.

Conclusion: The developed and validated method was found to be simple, accurate, economical, robust and reproducible. There was no interference of any diluent and excipient in the determination of drugs from synthetic mixture. So, the method can be successfully applied for routine Quality control analysis.

Keywords: Febuxostat, Naproxen, RP-HPLC, Synthetic mixture.

INTRODUCTION

Febuxostat (2-(3-cyano-4-isobutoxyphenyl)-4-methyl-1,3-thiazole-5-carboxylic acid) (ULORIC) (Figure 1) is a xanathine oxidase (XO) inhibitor indicated in patients with gout suffering from hyperuricemia and is used in its chronic management. Febuxostat received marketing approval by the European Medicines Agency on April 21, 2008 and was approved by the U.S. Food and Drug Administration on February 16, 2009. [1] It is freely soluble in Dimethylformamide; soluble in Ethanol; soluble in Methanol and Acetonitrile and practically insoluble in Water. [2] Naproxen(+)-(S)-2-(6-methoxynaphthalen-2-yl) propanoic acid (figure 2) is an anti-inflammatory agent with analgesic and antipyretic properties. Naproxen is commonly used for the reduction of pain, fever, inflammation and stiffness caused by conditions including migraine, osteoarthritis, kidney stones, rheumatoidarthritis,gout, ankylosingspondylitis, tendinitis.[1] Soluble in Water, Methanol and Ethanol, slightly soluble in Acetone, practically insoluble in Chloroform and Toluene. [2]

Fig. 1: Febuxostat [3]


Fig. 2: Naproxen [3]

Febuxostat is used as anti-gout.Naproxen is used as gout suppressant and pain killer. Combination of Febuxostat and Naproxen increases the Cmax and AUC Absorption of Febuxostat increases by 40%. Literature review revealed that there are several methods like U.V. Spectrophotometry [4-8], HPLC[9-31] avalilabe for estimation of Febuxostat and Naproxen as single formulation and in combination with other drugs. But literature review revealed that there is no method available for Simultaneous estimation of Febuxostat and Naproxen in combination. Thus, this article presents the simultaneous estimation of Febuxostat and Naproxen in synthetic mixture.

MATERIALS AND METHODS

Methanol HPLC grade and Acetonitrile HPLC grade was procured from E. Merck Ltd, Mumbai. Orthophosphoric acid, Potassium Dihydrogen phosphate was procured from Astron chemicals, Ahmedabad. The analysis of drug was carried out on a SHIMADZU LC2010 series HPLC system equipped with a reverse phase Hypersil C18 column (250×4.6mm, 5µm n particle size) a LC 20 AT isocratic pump, a 20µl injection loop and a SPD- 20 A Promenence UV- Visible Detector and running on Spinchrom Chromatographic Software version. Isocratic elution with 0.5 mM Phosphate buffer: Methanol: acetonitrile (ACN) (15: 35: 50 v/v) is used at a flow rate of 1 ml/ min. Milli-Q water is used for buffers and other reagents preparation.

HPLC conditions

The contents of mobile phase were Methanol, ACN, and phosphate buffer adjusted to pH 5 with 5% Orthophosphoric acid. These were filtered through 0.45µ membrane filter and degassed by sonication for use. The flow rate for mobile phase was optimized to 1ml/min.

The run time was set at 20 min and column temperature was maintain at ambient (room temperature). The volume of injection was 20 µL, and eluent was detected at 288 nm.

Method

The RP-HPLC method of Febuxostat and Naproxen were achieved by isocratic elution technique with UV-VIS Detector. Febuxostat and Naproxen were determined at 288nm respectively with the concentration range of 4-12 µg/ml for Febuxostat and 25-70 µg/ml for Naproxen respectively. For analysis of synthetic mixture, the powder of 10 mg of Febuxostat and 62.5 mg of Naproxen was taken, dissolved in 100 ml volumetric flask and made up to 100 ml with Methanol. The solution was sonicated for 15min and filtered through Whatmann filter paper No.41. From clear solution, further dilutions were made to get 10 µg/ml of Febuxostat and Naproxen.

Standard Stocks Solutions of binary mixture

About 10 mg of Febuxostat and 62.5 mg Naproxen weighed accurately and transferred to 100 ml volumetric flask and dissolved in methanol. This solution was sonicated for about 15 min and make up to the mark with methanol to get the concentration of 100 µg/ml Febuxostat and 625 µg/ml Naproxen.

Method validation

Specificity

A blank solution (mobile phase) and Common excipients like starch, lactose, magnesium stearate were dispersed in methanol, filtered and injected. The chromatogram showed no inferring peaks at retention time of the two drugs which indicates specificity of method.

Fig. 3: Chromatogram of mobile phase


Fig. 4: Chromatogram of excipients


Fig. 5: Chromatogram of binary mixture (nap-3.53 min and feb-4.83 min)


Table 1: Linearity data

Febuxostat Naproxen
Conc.(µg/ml) Mean Area± SD %RSD Conc.(µg/ml) Mean Area± SD % RSD
4 368594.3 ± 525.696 0.14 25 511140.3 ± 1090.314 0.21
6 421046.3±1161.009 0.27 32.5 572052.3 ± 3107.962 0.54
8 473462.3 ± 1077.19 0.22 50 638483.7 ± 5802.145 0.90
10 527129.3 ±1776.04 0.33 62.5 705566.3 ± 5342.425 0.75
12 579406 ±1620.786 0.27 75.0 771264.7 ± 7397.649 0.95

Linearity and range

Aliquots of standard stock solutions of Febuxostat and Naproxen were taken in 10 ml volumetric flasks and diluted with mobile phase to get final concentrations in range of 4-12μg/ml for Febuxostat and 25-75μg/ml for Naproxen. Triplicate injections were made for each concentration.

Fig. 7: Febuxostat (4-12 μg/ml)

The plots of peak area versus respective concentrations of Febuxostat and Naproxen were found to be linear in the concentration range of 4-12μg/ml and 25-75μg/ml respectively. The linear regression equations of the lines are:

For Febuxostat -y = 26375x + 262916, (r2 = 1)

For Naproxen -y = 5250.8x + 377336, (r2 = 0.9998)

For Febuxostat

Precision

Precision study was performed as per ICH Q2 (R1)[32] to find out repeatability, intra-day and inter-day variations. For the precision, three replicates of each concentration are injected into the system and results are reported in terms of relative standard deviation as given in table No. 2,3and 4 respectively.

Table 2: Repeatability data

Drug Name Febuxostat Naproxen
Wavelength 288 nm
Concentration 8 µg/ml 50 µg/ml
MEAN±SD(n=3) 550955.8 ± 940.8129 723179.3 ± 1961.269
%RSD 0.17 0.27

Fig. 8: Naproxen (25-75 μg/ml)

Accuracy

The accuracy was determined by recovery studies. The recovery studies were performed by standard addition method, at 80%, 100%, 120% level. Percent recovered was calculated using regression equation.

Limit of Detection and Limit of Quantitation

The limit of detection (LOD) is the smallest concentration that can be detected but not necessarily quantified as an exact value.

LOD = 3.3 × Standard deviation of y intercept /Slope of calibration curve

Febuxostat – 0.105μg/ml

Naproxen – 1.93μg/ml

The LOQ is the lowest amount of analyte in the sample that can be quantitatively determined with suitable precision and accuracy.

LOQ = 10 × Standard deviation of y intercept/ Slope of calibration curve

Febuxostat – 0.316μg/ml

Naproxen – 5.86μg/ml

Applicability to synthetic mixture

Applicability of proposed method was tested by analyzing the synthetic mixture.

Table 3: Intraday precision data

Febuxostat
Wavelength = 288 nm

Naproxen
Wavelength = 288 nm

Conc.
(µg/ml)

Mean Area ± SD

% RSD

Conc.
(µg/ml)

Mean Area ± SD

% RSD

6

441926.3 ± 619.1545

0.14

37.5

591421.3 ± 282.2788

0.04

10

552418.3 ± 711.5703

0.12

62.5

725942 ± 233.8269

0.03

14

661356.7 ± 807.8814

0.12

87.5

858148.3 ± 2791.556

0.3

Table 4: Interday precision data

Febuxostat
Wavelength = 288 nm

Naproxen
Wavelength = 288 nm

Conc.
(µg/ml)

Mean Area ± SD

% RSD

Conc.
(µg/ml)

Mean Area ± SD

% RSD

6

435200.3 ± 11232.98

1.39

37.5

584190.7 ± 2263.98

1.46

10

543938 ± 14944.42

0.72

62.5

717163.7 ± 5088.96

0.69

14

651883 ± 16758.17

0.99

87.5

851619.3 ± 8517.86

0.81

Table 5: Accuracy data

Drug name % Level of Recovery Total amount (µg/ml) Total amount obtained(n=3) ± SD Area± SD % Recovery ± SD
Feb 80 % 7.2 7.1±0.03 409975±139.0 99.5 %±0.0577
100 % 8.0 8.0±0.04 457843±724.6 100.8 %±0.12
120 % 8.8 8.7±0.01 494441±1460 99.59 % ±0.31
Nap 80 % 45 44.7±0.43 464680±3766 99.41 % ±0.91
100 % 50 50.9±0.51 516868±4662 100 %±1.02
120 % 55 55.26±0.80 555129.3±731 99.52%±1.46

Fig. 6: Chromatogram of synthetic mixture


Table 7: Applicability to synthetic mixture Data

Synthetic mixture Drug Amount taken in synthetic mixture Amount found (mg) (n=3) ± SD. %Amount obtained ± SD.
Febuxostat 40 mg 39.8 ± 0.52 99.5% ± 0.125
Naproxen 250 mg 251.0 ± 0.763 100.4% ± 1.014

RESULT


Table 8: Optical regression characteristics and validation parameters

S. No. Parameters Febuxostat Naproxen
1 Wavelength (nm) 288 nm
2 Beer’s Law Limit (μg/ml) 4 – 12 25 – 75
3 Regression equation(y = mx +c) y = 26375x + 262916 y = 5250.8x + 377336
4 Slope (m) 26375 5250.8
5 Intercept (c) 262916 377336
6 Correlation Coefficient (r²) 1.0 0.999
7 Repeatability (% RSD, n=6) 0.17 0.27
8 Interday (n=3) (% RSD) (μg/ml) 1.5 – 1.7 1.0 – 2.0
9 Intraday(n=3) (% RSD) (μg/ml) 0.12 – 0.14 0.03 – 0.3
10 LOD(μg/ml) 0.105 1.93
11 LOQ(μg/ml) 0.319 5.86
12 Accuracy 99.5-100.8 % 99.4-100 %

CONCLUSION

A Simple, specific, accurate, and precise RP-HPLC method has been developed and validated for simultaneous estimation of both these drugs. The chromatographic separation was achieved on Hypersil BDS coloumn (250 mm x 4.6mm, 5μm) in size column using ACN: Methanol: Phosphate Buffer (50:35:15 v/v) as mobile phase at 288 nm.

The plot of area versus respective concentration of Febuxostat and Naproxen was found to be linear in the concentration range of 4-12 μg/ml for Febuxostat and 25- 75 μg/ml for Naproxen with r2 = 1 for Feb and r2 = 0.998 for Nap respectively.

Precision was calculated as repeatability, interday and intraday variations and % RSD was found to be less than 2% for both the drugs.

Mean % recovery for Febuxostat and Naproxen were found to be 100.8 % and 99.52 % respectively. Recovery is in the range of 98-102 %, indicates that method is accurate.

System suitability test reveals that all system suitability parameters complies standard values.

CONFLICT OF INTERESTS

Declared None

ACKNOWLEDGEMENT

The authors are thankful to Dr. Kilambi Pundarikakshudu, Director of L.J. Institute of Pharmacy, Sarkhej, Ahmedabad who provided me all facilities and good faculty in my college.I am thankful to AMI Life science Ltd, Baroda and Intas Pharma Ahmedabad for providing drug samples Febuxostat and Naproxen for research.

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