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1 International Journal of Universal Pharmacy and Bio Sciences 2(3): May-June 2013 INTERNATIONAL JOURNAL OF UNIVERSAL PHARMACY AND BIO SCIENCES Pharmaceutical Sciences Research Article!!! Received: ; Accepted: DEVELOPMENT AND VALIDATION OF STABILITY INDICATING RP-HPLC METHOD FOR ESTIMATION OF BROMFENAC SODIUM IN THE PHARMACEUTICAL FORMULATION Meha Patel*, Manisha Gohil, Shailesh Koradia Babaria Institute of Pharmacy BITs Edu Campus Vadodara-Mumbai NH#8, Varnama, Vadodara, Gujarat, India. KEYWORDS: Bromfenac Sodium, Stability indicating RP- HPLC Validation Studies. Method, For Correspondence: Meha Patel* Address: Babaria Institute of Pharmacy BITs Edu Campus Vadodara-Mumbai NH#8, Varnama, Vadodara, Gujarat, India. Mb No: ABSTRACT The simple, accurate and precise stability indicating RP-HPLC methods have been developed for the estimation of Bromfenac sodium in the pharmaceutical formulation. In the stability indicating RP-HPLC method, the chromatographic separation was performed on Hypersil C 18 column (150 mm 4.6 mm, i. d., 5 μm). Mobile phase consisted of a mixture of acetonitrile, 0.025M sodium dihydrogen orthophosphate buffer ph 4.0 and triethylamine in the ratio of 70:30:0.2, v/v/v at a flow rate of 1.0 ml/min. The detection wavelength was set at 270 nm. The degradation products of Bromfenac sodium were completely separated. The linearity and correlation coefficient was found to be µg/ml and for Bromfenac sodium, respectively. The percentage recovery and %RSD was found to be 99.76% and for Bromfenac sodium respectively. The method was validated as per ICH guidelines and successfully applied to the determination of Bromfenac sodium in the quality control laboratory. 438

2 INTRODUCTION: Bromfenac sodium has anti-inflammatory activity thought to be due to its ability to block prostaglandin synthesis by inhibiting COXs, an important group of enzymes active in the inflammatory process, which catalyzes the biosynthesis of eicosanoids from arachidonic acid to produce PGs and thromboxanes. Prostaglandins are mediators of certain kinds of intraocular inflammation. In studies of animal eyes, PG has been shown to disrupt the blood aqueous humor barrier and increase vascular permeability, vasodilatation, intraocular pressure, and leukocytosis. Br O NH 2 CH 2 CO 2 Na.1 1/2 H 2 O MATERIALS AND METHODS: Figure 1: Structure of Bromfenac sodium Reagents All chemicals and reagents used were of a HPLC grade or an analytical grade. Bromfenac sodium bulk powder was kindly gifted by Enaltec laboratory Pvt. Ltd. Mumbai, Maharashtra (India). Methanol, Acetonitrile, Water, Orthophosphoric acid, Tri ethylamine, sodium dihydrogen phosphate, Nylon filter paper. Equipment HPLC Instrument Model : Hitachi HPLC system Column : ODS Hypersil C 18 [250 mm x 4.6mm, 5μm] Pump : L-2130 Detector : L-2400 Data processor : EZ Start A double beam UV-visible Spectrophotometer (Shimadzu, UV-1800), attached to a computer software UV probe 2.34, with a spectral width of 2 nm, wavelength accuracy of 0.2 nm and pair of 1 cm matched quartz cells. Electronic analytical Weighing balance: Shimadzu (AUX220) Digital ph meter: Toshcon Industries Pvt. Ltd. Ultrasonic cleaner: EIE 808, EIE Instruments Pvt. Ltd Volumetric flasks, Pipettes & Funnel All the instruments and glass wares were calibrated prior to use. 439

3 Chromatographic conditions The column used was ODS Hypersil C 18 [250 mm x 4.6 mm, 5μm] for chromatographic separations. The chromatographic separation was carried out under isocratic elution with ACN/0.025 M Sodium dihydrogen orthophosphate buffer (ph 4.0 ± 0.1), 70:30 (v/v). The mobile phase was filter through 0.45µm membrane filter and degassed by ultra sonic bath. The mobile phase was pumped at a flow rate of 1.0mL/min at ambient temperature, the UV detection was carried out at 270nm and the injection volume was 20µL. Preparation of mobile phase Sodium dihydrogen orthophosphate (3.9 g) was accurately weighed, dissolved in 1000mL volumetric flask with water and adjusted ph 4.0 with orthophosphoric acid. The mobile phase consisting of ACN/0.025M buffer (70:30 v/v) was filtered through 0.45µm membrane filter and degassed prior to use. Preparation of standard stock solution of BROM Accurately weighed quantity of about 25 mg of BROM was transferred into a 25 ml volumetric flask. It was dissolve and dilute to the mark with distilled water to obtained 1000 µg/ml. An aliquot of 2.5 ml of stock solution was transferred into 25 ml volumetric flask and dilute to the mark with distilled water to obtained 100µg/mL. This solution was used as working standard solution. Preparation of sample solution of BROM Accurately measured volume of 5 ml of sample (label claim 0.9 mg/ ml) was transferred into a 50 ml volumetric flask and diluted to the mark with distilled water to obtained 90 µg/ml. Selection of wavelength The standard solutions of BROM (20 µg/ml) in mobile phase were scanned separately in the UV region of nm using mobile phase as a blank. From the spectrum, 270 nm was selected as detection wavelength as drugs exhibit sufficient absorptivity at this wavelength (Figure 2). Stability of solution Sample solution was kept at room temperature (24 hours) and 2-8 C (4 days), respectively. The assay of initial time period was compared with these two time periods. The difference between assays should not be more than 2.0% for solution. METHOD VALIDATION System suitability An aliquot of 5.0 ml (100 μg/ml) standard stock solution of BROM was transferred into a 10 ml volumetric flask and diluted upto mark with mobile phase. 20 µl of this standard solution BROM (50 μg/ml) solution was injected in to HPLC system. Retention time, peak area, theoretical plates and tailing factor in chromatogram for BROM was observed and were presented in Fig.3 & Tab

4 Specificity Specificity of an analytical method is ability to measure specifically the analyte of interest without interferences from blank and placebo. For the determination of specificity 50μg/ml solution of the standard BROM, and the same diluted solution of placebo were injected and chromatographs were recorded and observed for any interference of placebo. Placebo spectra must not show any significant absorbance at the retention time of BROM. (Figure 3, 4, 5) Calibration curve (Linearity) An aliquots of 1.0 ml, 2.0 ml, 3.0 ml, 4.0 ml, and 5.0 ml of 100µg/mL of standard stock solution of BROM were transferred into separate 10 ml volumetric flasks and diluted upto the mark with mobile phase to obtain the concentrations of 10 μg/ml, 20 μg/ml, 30 μg/ml, 40 μg/ml and 50 μg/ml respectively.20 μl were injected to get the concentration range of μg/ml of BROM. Calibration curve was constructed by plotting (peak areas v/s concentration) of BROM.The results are presented in (Table 2 and Figure 6). Accuracy (% Recovery) Level I: An aliquot of 2.2 ml of sample stock solution and 1.8 ml of 100 μg/ml standard stock solutions of BROM was transferred into 10 ml volumetric flask and diluted upto the mark with mobile phase. Level II: An aliquot of 2.2 ml sample stock solution and 2.0 ml of 100 μg/ml standard stock solutions BROM was transferred into 10 ml volumetric flask and diluted upto the mark with mobile phase. Level III: An aliquot of 2.2 ml sample stock solution and 2.2 ml of 100μg/mL standard stock solutions of BROM was transferred in 10 ml volumetric flask and diluted upto the mark with mobile phase. 20 μl of solution of each resulting mixtures were injected repeatedly into the system and from chromatogram, retention time and peak area were recorded and the result are presented in Table 4. Repeatability An aliquots of 1.0 ml of 100 µg/ml of standard stock solution of BROM was transferred to a separate 10 ml of volumetric flasks and diluted upto the mark with mobile phase to obtain a concentrations of 10 μg/ml. Repeatability was performed six times of the same solution. The results are presented in terms of % relative standard deviation (% RSD) in Table 6. Intraday and Interday precision An aliquots of 1.0 ml, 3.0 ml and 5.0 ml of 100 µg/ml of standard stock solution of BROM were transferred to a separate 10 ml of volumetric flasks and diluted upto the mark with mobile phase to obtain a concentrations of 10 μg/ml, 30 μg/ml and 50 μg/ml for BROM. 20 μl of each of these standard solutions BROM was injected under the operating chromatographic conditions 441

5 into system three times on the same day and chromatograms were recorded. Interday precision was evaluated by using freshly prepared solution of BROM (10, 30, 50 μg/ml) in three different days as per proposed method. The results are presented in terms of % relative standard deviation (% RSD) in Table 3. Limit of detection and Limit of quantitation LOD and the LOQ of the drug were calculated using the following equations as per International Conference on Harmonization (ICH) guidelines. LOD = 3.3 σ/s LOQ = 10 σ/s Where, σ = Standard deviation of the response S = Slope of calibration curve. The results of LOD and LOQ are presented in Table 6. Forced degradation studies of BROM In order to establish stability-indicating nature of the method, pure Bromfenac sodium (API), and pharmaceutical formulation were subjected to various stress conditions to conduct forced degradation studies. Standard stock solution is used for forced degradation studies. Stress studies were carried out under the conditions of acid/base hydrolysis, oxidation, thermal, UV light as mentioned in ICH Q1A (R2) guideline. Preparation of acid induced degradation product To 100 µg/ml of stock solution (5.0 ml) of BROM and sample solution 90 µg/ml (3.3mL) was transferred in 10ml volumetric flask, in acidic media was performed by keeping 1 ml of solution in contact with 1 ml of 2 N HCl and heated for up to 2 hrs at 80 C. The solution was neutralized with 2 N NaOH and diluted with mark with mobile phase to get final concentration of 50 µg/ml BROM and 30 µg/ml of sample respectively. Preparation of base induced degradation product To 100 µg/ml of stock solution (5.0 ml) of BROM and sample solution 90 µg/ml (3.3mL) was transferred in 10ml volumetric flask separately, in basic media was performed by keeping 1 ml of solution in contact with 1 ml of 2 N NaOH and heated for up to 2 hrs at 80 C. The solution was neutralized with 2 N HCl and diluted upto mark with mobile phase to get final concentration of 50 µg/ml BROM standard and 30 µg/ml of sample respectively. Preparation of H 2 O 2 induced degradation product To 100 µg/ml of stock solution (5.0 ml) of BROM and sample solution 90 µg/ml (3.3 ml) was transferred in 10ml volumetric flask separately, in oxidative degradation was performed by adding 1 ml of 6% hydrogen peroxide in flask. 442

6 These mixtures was kept for 2 hrs in room temperature and diluted upto mark with mobile phase to get final concentration of 50 µg/ml BROM standard and 30 µg/ml of sample respectively. Preparation of thermal degradation product To 100 µg/ml of stock solution (5.0 ml) of BROM and sample solution 90 µg/ml (3.3mL) was transferred in 10ml volumetric flask, diluted upto mark with mobile phase to get final concentration of 50 µg/ml BROM standard and 30 µg/ml of sample were keep solution to 80 C in Oven for 2 hrs. Preparation of photo degradation product To 100 µg/ml of stock solution (5.0 ml) of BROM and sample solution 90 µg/ml (3.3mL) was transferred in 10ml volumetric flask, diluted upto mark with mobile phase to get final concentration of 50 µg/ml BROM and 30 µg/ml of sample were keep solution at UV light for 2 hrs in UV chamber at 254 nm. RESULTS AND DISCUSSION SELECTION OF WAVELENGTH Figure 2: Spectrum of BROM (20µg/mL) SPECIFICITY There was no interference of placebo at the retention time of standard BROM as shown in figure 3, 4, 5. Figure 3: Chromatogram of mobile phase (Blank) 443

7 Figure 4: Chromatogram of standard BROM (50µg/mL) (t R= 5.310) at 270 nm Figure 5: Chromatogram of sample BROM (30µg/mL) (t R= 5.587) at 270 nm SYSTEM SUITABILITY This parameter ensures that the analytical system is working properly and can give accurate and precise results. A 20 μl solution of BROM (50μg/ml) was injected and the parameter like theoretical plates per column, tailing factor were calculated from the chromatogram. Various system suitability parameters for the developed method are shown in Table 1. Table 1: System suitability parameters Parameter BROM Acceptance criteria Retention time 5.31 ± 0.5 (min) - Theoretical Plates ± SD 3589 ± NLT 2000 asymmetry factor ± SD ± NMT 2 Capacity factor ± NLT 2 CALIBRATION CURVE (LINEARITY) The linearity of the response of the drug was found to be between μg/ml concentrations. The calibration graphs were obtained by plotting the peak area versus the concentration data and were treated by linear regression analysis (Table2). 444

8 The equation of the calibration curve for BROM obtained y = 222,565.61x - 99,606.51, the calibration curve was found to be linear in the aforementioned concentrations. The correlation coefficient (r 2 ) of determination was Table 2: Calibration data of BROM Concentration (µg/ml) Peak area mean ± SD (n=3) ± ± ± ± ± Figure 6: Calibration curve of Bromfenac sodium PRECISION Method precision (intra-day precision) was evaluated by carrying out three independent measurements of standard drug solution at three concentrations of BROM three times on the same day. The relative standard deviation (RSD) values obtained for three concentrations (10μg/mL, 30μg/mL and 50 μg/ml) at intraday precision were , and respectively (Table3). Mean % RSD was found to be All the data are within the acceptance criteria of 1%. Table 3: Interday and Intraday precision of BROM Concentration (µg/ml) BROM Peak area Mean ± SD (n=9) %RSD Interday Intraday Interday Intraday ± ± ± ± ± ± Mean

9 ACCURACY (RECOVERY) The recovery was performed by preparing concentration of 90 μg/ml of BROM sample solution. Three samples were prepared for each recovery level of 80%, 100% and 120% spiking of standard solution. The solutions were then analyzed, and the percentage recoveries were calculated from the calibration curve. The accuracy was calculated as the percentage of the drug recovered from the formulation matrix. The equation used for the calculation was as follows: % Recovery = [(Spiked area unspiked area) / standard area] x 100 The mean percentage recovery obtained was ± which is presented in Table 4 and was considered acceptable. Table 4: Accuracy (Recovery) data of BROM by proposed HPLC method Level of Recovery Amount of drug taken (µg/ml) Amount of drug added (µg/ml) Amount of drug recovered (n=9) %Recovery ± SD %RSD 80% ± % ± % ± LOD & LOQ The peak area of six solutions containing 10 µg/ml were measured at 270 nm and calculated according to equation of LOD [3.3 x MSD/ slope] and LOQ [10 x MSD/ slope]. The values were μg/ml and μg/ml respectively (Table 6). ASSAY OF PHARMACEUTICAL FORMULATION Bromfenac sodium eye drops each ml contain 0.9mg of Bromfenac sodium, so 5ml Bromfenac sodium eye drops contain 4.5mg of Bromfenac sodium and transfer into a 50 ml volumetric flask. Make up the volume with mobile phase (90µg/ml). Take 3.3 ml solution in 10 ml volumetric flask make up the volume with mobile phase. After setting the chromatographic conditions and stabilizing the instrument to obtain a steady baseline, the sample solution was injected, chromatogram was obtained and the peak areas were recorded. The injections were repeated six times and the amount of each drug present in sample was estimated from their respective calibration curve. Table 5: Assay of pharmaceutical formulation Formulation Label value (mg/5ml) Amount found (mg/5ml) % Drug found Mean SD (n=6) %RSD Eye drops 4.50mg 4.44mg ±

10 SUMMARY OF THE OBTAINED RESULTS Table 6: Regression analysis data and summary of validation parameters Sr. No. Parameter Results 1 Absorption maxima (nm) Linearity range (μg/ml) Standard Regression equation y = 222,565.61x - 99, Correlation coefficient (R 2 ) LOD (μg/ml) LOQ (μg/ml) % Recovery (Accuracy, n = 9) ± Precision (%RSD) Repeatability(n=6) Forced degradation studies Acid degradation Intraday (n= 9) Interday (n= 9) Figure 7: Chromatogram of acid treated of 2N HCl (Blank) Figure 8: Chromatogram of acid treated standard BROM (2N HCl, and heated for up to 2 hrs at 80 C) 447

11 Figure 9: Chromatogram of acid degraded BROM sample (2N HCl, and heated for up to 2 hrs at 80 C) Base degradation: Figure 10: Chromatogram base treated of 2N NaOH (Blank) Figure 11: Chromatogram of base degraded standard BROM (2N NaOH, and heated for up to 2 hrs at 80 C) 448

12 Figure 12: Chromatogram of base degraded sample BROM (2N NaOH, and heated for up to 2 hrs at 80 C) Oxidative degradation Figure 13: Chromatogram of 6% H 2 O 2 treated (Blank) Figure 14: Chromatogram of 6% H 2 O 2 treated standard BROM 449

13 Thermal degradation Figure 15: Chromatogram of 6% H 2 O 2 treated sample BROM Figure 16: Chromatogram of thermal degradation standard BROM Figure 17: Chromatogram of thermal degradation sample BROM 450

14 Photo degradation International Standard Serial Number (ISSN): Figure 18: Chromatogram of photo degradation of standard BROM Figure 19: Chromatogram of photo degradation sample BROM Table 7: Summary of degradation studies for BROM standard and sample Degradation condition Acid, 2N HCl (heated at, 80 o C) Base, 2N NaOH(heated at, 80 o C) Time % Degradation R T (min) of degradation product API Sample API Sample BROM BROM BROM BROM DP1 DP 2 DP1 DP2 2 hrs 14.40% 15.06% hrs 15.09% 14.97% Oxidation, 6% 2 hrs 13.55% 11.98% H 2 O 2 Thermal 2 hrs 11.86% 10.07% (80 o C) UV light, 2hrs 12.40% 12.03% nm *BDP1, 2 = Brom degradation product 451

15 CONCLUSION International Standard Serial Number (ISSN): The degradation products of Bromfenac sodium were completely separated. The linearity and correlation coefficient was found to be µg/ml and for Bromfenac sodium, respectively. The percentage recovery and %RSD was found to be 99.76% and for Bromfenac sodium respectively. The method was validated as per ICH guidelines and successfully applied to the determination of Bromfenac sodium in the quality control laboratory. ACKNOWLEDGEMENT The authors are highly thankful to Babaria Institute of Pharmacy, Varnama, Vadodara, Gujarat, India for providing all the facilities to carry out the work. REFERENCES: European medicines agency, science medicines health, committee for medicinal product for human use (CHMP) 19 may (2011) 5. Beckett, A. H., Stenlake, J. B., (1997), Practical Pharmaceutical Chemistry, Part-II, 4 th Edn., CBS Publishers and distributors, India. 6. ICH, Q2 (R1) Validation of Analytical Procedure, Text and Methodology, International Conference on Harmonization (2005). 7. Ahuja S., Stephen S., Modern Pharmaceutical analysis, separation science and technology, vol.3, Reynolds DW, Fachine KL, Mullaney JF, Alsante KM, Hatajik TD, Motto MG. (2002), Available guidance and best practices for conducting forced degradation studies. Pharm Tech; 26, Walsh DA, Moran H, (1984), "Anti-inflammatory agents-synthesis and pharmacological evaluation of 2-amino-3 benzoylphenylacetic acid and analogs, Journal of Medicinal Chemistry, 27(11), Parmar AN., Parmar RR, Patel VM. Shah DA. (2012), The Simultaneous Estimation of Moxifloxacin Hydrochloride and Bromfenac sodium in Eye Drops by UV. JPSBR: Volume 2, Vyas PJ., Dave JB., Patel CN.; (2012), Simultaneous Estimation of Moxifloxacin HCL and Bromfenac sodium in eye drops by spectrophotometric methods. IJPSR, vol.3(7)

16 13. ICH guidelines Q1A (R2). Stability Testing of New Drug Substances and Products (revision 2); November (2003) 14. Sethi PD. (2001), HPLC Quantitative analysis of pharmaceutical formulations. CBS Publication and Distributors, New Delhi, 1-20, Kan J, Biao WU, Hong- Xiao CUI, (2009), RP-HPLC determination of content and related substances of Bromfenac sodium, Chinese Journal of Pharmaceutical Analysis,

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