DEVELOPMENT AND VALIDATION OF FIRST ORDER DERIVATIVE UV-SPECTROSCOPIC METHOD FOR ESTIMATION OF IBUPROFEN AND FAMOTIDINE IN SYNTHETIC MIXTURE

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1 PHARMA SCIENCE MONITOR AN INTERNATIONAL JOURNAL OF PHARMACEUTICAL SCIENCES DEVELOPMENT AND VALIDATION OF FIRST ORDER DERIVATIVE UV-SPECTROSCOPIC METHOD FOR ESTIMATION OF IBUPROFEN AND FAMOTIDINE IN SYNTHETIC MIXTURE Deep P. Patel*, Ragin R. Shah, Alisha P. Patel and Ponal K. Tank Department of Quality Assurance, Arihant School of Pharmacy & BRI, Adalaj, Gandhinagar , Gujarat, India. ABSTRACT In the present work, sensitive spectrophotometric method has been developed for the quantitative estimation of Famotidine (FAM) and Ibuprofen (IBU) individually as well as in combined dosage form. Methanol was selected as a common solvent for estimation of both the drugs. λ max for FAM and IBU were found to be 285 nm and 263 nm respectively. First order derivative spectrophotometric method involves the measurement of absorbance of one drug at zero crossing point of other drug, 249 nm and nm was selected for the estimation of IBU and FAM respectively. Linear range was found 4-20 μg/ml and μg/ml with (r 2 = , % CV = 0.32 to 1.49 and r 2 = , % CV = 0.71 to 1.50) for FAM and IBU respectively. LOD for FAM and IBU were found to be μg/ml and10 μg/ml respectively and LOQ for FAM and IBU were found to be μg/ml and 30 μg/ml respectively. The method was validated as per the International Conference on Harmonization (ICH) guidelines. The proposed validated method was successfully used for the quantitative analysis of synthetic mixture. Keywords: Famotidine, Ibuprofen, First order derivative spectroscopy, ICH. INTRODUCTION Famotidine, chemically 3-[({2-[(diaminomethylidene) amino] -1, 3- thiazol- 4-yl} methyl) sulfanyl]-n'-sulfamoylpropanimidamide [1]. It is a competitive histamine H 2 receptor antagonist. Its main pharmacodynamics effect is the inhibition of gastric secretion [2]. Ibuprofen, chemically 2-[4-(2-methylpropyl) phenyl] propanoic acid [3]. It is a propionic acid derivative is a prototypical non-steroidal anti-inflammatory agent (NSAIA) with analgesic and antipyretic properties [4]. IC Value

2 Famotidine Ibuprofen Famotidine and Ibuprofen both are official in IP [5], USP [6], BP [7], JP 15 [8] and EP [9] which includes HPLC method, Potentiometric titration for estimation of FAM and IBU respectively. Famotidine and Ibuprofen are available in a combined tablet dosage named DUEXIS (Horizon Pharma). It was approved by US-FDA. The combination of these two drugs is not official in any pharmacopoeia. Literature review [10-14] shows that numbers of analytical methods are available for estimation of both the drugs either alone or in combination with other drugs. Based on our current and ongoing referencing work, till date, we have not come across any official and reported analytical methods for simultaneous estimation of both the drugs in their combined dosage form. Therefore, the objective is to develop a spectroscopic method for simultaneous estimation of Famotidine and Ibuprofen in synthetic mixture and to validate the developed method according to ICH guidelines [15]. MATERIALS AND METHODS Instruments: Shimadzu UV-1800, UV-Visible double beam Spectrophotometer with matching pair of 1 cm quartz cuvettes (Shimadzu Corporation, Kyoto, Japan). The spectral bandwidth is 0.5 nm. Electronic analytical balance (Shimadzu AUX-220) was used in study. Reagents and Chemicals: Standard Famotidine (FAM) and Ibuprofen (IBU) were kindly gifted by Vaibhav Analytical Laboratories, Ahmedabad, India. Methanol (A.R. Grade - Chemco Chemicals IC Value

3 Ltd.) was used in study. Microcrystalline cellulose (MCC), Crosscarmellose sodium (CCS), PVP K30, Magnesium stearate, Talc, Distilled water. Methodology (First order derivative method): Preparation of solutions for Famotidine and Ibuprofen for determination of wavelength maxima (λ max ) and zero crossing point: Accurately weighed FAM (25.0 mg) and IBU (750.0) and transferred to a 25 ml volumetric flask and dissolved and diluted up to the mark with methanol to produced 1000 μg/ml for FAM and μg/ml for IBU stock solution. Further dilution was made to produce concentrations of 100 μg/ml FAM and 3000 μg/ml for IBU (SS1). From this diluted solution further dilution was made to get the concentrations of FAM in range of 4-20 μg/ml and for IBU in range of μg/ml. Each of the solution was scanned between nm at a medium scanning speed. It showed wavelength maxima at 285 nm for FAM and 263 nm for IBU. All the Zero order overlain spectra were then converted to their respective first order Derivative Spectra using the inbuilt software and Zero Crossing point (ZCP) of FAM and IBU were found to be at 249 nm and nm respectively. Responses of each of the above solutions were measured at 249 nm (ZCP of FAM) and nm (ZCP of IBU). Lambert Beer s curves were plotted for IBU and FAM at 249 nm and nm respectively. The straight line equations and correlation coefficients for FAM and IBU were determined. Method validation: Linearity and Range (n = 5): The linearity response was determined by analyzing 5 independent levels of calibration curve in the range of 4-20 µg/ml (4, 8, 12, 16 and 20 μg/ml) for FAM and µg/ml (120, 240, 360, 480 and 600 μg/ml) for IBU. The calibration curve of responses against concentration was plotted. Correlation coefficient and regression line equations for IBU and FAM were calculated. Linearity range was established through consideration of necessary practical range and according to each drug concentration present in the pharmaceutical product, to give accurate, precise and linear results. Accuracy (n = 3): IC Value

4 It was carried out to determine the suitability and reliability of the proposed method. Accuracy was determined by calculating the % Recovery of FAM and IBU from the Synthetic mixture by the standard addition method in which, known amounts of standards samples of FAM and IBU at 50%, 100% and 150% levels were added to the pre-analysed samples. The recovered amounts of FAM and IBU were calculated at each level and % Recovery was reported. Precision: Repeatability (n = 6): For the repeatability study, From the SS1, aliquot of 1.2 ml was transferred to a separate 10 ml volumetric flask and diluted up to mark with methanol such that it gives the concentration of 12 µg/ml of FAM and 360 µg/ml of IBU. The absorbance of the solution was measured at nm and 249 nm. The procedure was repeated six times and % CV was calculated. Intraday Precision (n = 3): From the SS1, aliquots of 0.4 ml, 1.2 ml and 2.0 ml were transferred to separate 10 ml volumetric flasks and diluted up to the mark with methanol to give the concentration of 4, 12 and 20 µg/ml for FAM and 120, 360 and 600 µg/ml for IBU. The solutions were analyzed three times on the same day and % CV was calculated. Interday Precision (n=3): From the SS1, aliquots of 0.4 ml, 1.2 ml and 2.0 ml were transferred to separate 10 ml volumetric flasks and diluted up to the mark with methanol to give the concentration of 4, 12 and 20 µg/ml for FAM and 120, 360 and 600 µg/ml for IBU. The solutions were analyzed on three different days and % CV was calculated. LOD and LOQ: The LOD and LOQ were estimated from the set of 5 calibration curves. They were calculated as, LOD = 3.3 (SD/Slope) and LOQ = 10 (SD/Slope) Where, SD = Standard deviation of the Y- intercepts of the 5 calibration curves. Slope = Mean slope of the 5 calibration curves. Estimation of Famotidine and Ibuprofen in the synthetic mixture: IC Value

5 Prepare synthetic mixture: Accurately weight 25 mg of FAM and 750 mg of IBU and transfer it in to a mortar then add mg of MCC (LR Grade), 3% Crosscarmellose sodium (LR Grade), 1.6% PVP K30 (LR Grade), 1.5% Mg. stearate (LR Grade) and finally 1.5% of Talc (LR Grade). Mix it in mortar for 15 min. Above mixture was transferred to 25 ml volumetric flask and dissolved in sufficient quantity of methanol. The contents were ultrasonicated for 10 min and final volume made up with methanol. The solution was than filtered through Whatman filter paper (No. 41). From above solution 1.0 ml aliquots of the solution was taken and transferred to 10 ml volumetric flask. Volume was made up to the mark with the methanol to produce solution containing FAM (100 μg/ml) and IBU (3000 μg/ml). From this solution 0.4 aliquots of the solution was taken and transferred to 10 ml volumetric flask. Volume was made up to the mark with the methanol to produce solution containing FAM (12 μg/ml) and IBU (360 μg/ml). This solution was used for the estimation of FAM and IBU. The responses of the solution were measured using first order derivative spectrophotometry at the ZCP of FAM (249 nm) for estimation of IBU and at the ZCP of IBU (263.6 nm) for estimation of FAM. The concentration of each drug was calculated using equation of regression line. RESULTS AND DISCUSSION The wavelength maxima for FAM and IBU were found to be 285 nm and 263 nm respectively using methanol as a solvent (Fig. 1 and Fig. 2). The ZCPs for FAM and IBU were found to be 249 nm and nm respectively (Fig. 3). Results of the validation of the above method indicate that the method was linear in the range of 4-20 μg/ml for FAM and μg/ml for IBU (Table 1 and Table 2, Fig. 4 and Fig. 5). The data for all validation parameters are mentioned in Table 4. The % recoveries for FAM and IBU obtained in the accuracy study were % and % respectively. The results of the precision study indicate that the proposed method showed good repeatability for FAM and IBU with % CV of 0.32 and 0.51 respectively. The % CV for Intraday precision was found to be for FAM and for IBU. Similarly % CV from the interday precision data were found be found to be for FAM and for IBU. The LOD for FAM and IBU was found to be IC Value

6 µg/ml and 10 µg/ml respectively. Similarly LOQ for FAM and IBU was found to be µg/ml and 30 µg/ml respectively. λ max = 285 nm λ max = 263 nm Figure 1 Zero order overlain spectra of Famotidine (4 20 µg/ml) Figure 2 Zero order overlain spectra of Ibuprofen ( µg/ml) ZCP OF FAMO = 249 nm ZCP OF IBU = 263.6nm Figure 3 Overlain 1 st order derivative spectra of Famotidine and Ibuprofen IC Value

7 TABLE 1: LAMBERT-BEER S CURVE DATA FOR IBUPROFEN AT 249 NM Conc. (μg/ml) Response % CV Mean ± S.D ± ± ± ± ± Figure 4 Calibration curve for Ibuprofen ( μg/ml) Figure 5 Calibration curve for Famotidine (4-20 μg/ml) TABLE 2: LAMBERT-BEER S CURVE DATA FOR FAMOTIDINE AT NM Conc. Response % CV (μg/ml) Mean ± S.D. (n=5) ± ± ± ± ± TABLE 3: DATA OF REGRESSION ANALYSIS OF FAMOTIDINE AND IBUPROFEN Straight line equation of Correlation Drug Calibration curve coefficient IBU Y = x FAM Y = x IC Value

8 TABLE 4: SUMMARY OF VALIDATION PARAMETERS PARAMETER FAM IBU Linearity 4-20 μg/ml μg/ml Accuracy (n=3) % % Precision Repeatability (%CV, n=6) Intraday (n=3) Interday (n=3) LOD LOQ Assay 99.52± ± DRUG TABLE 5: ANALYSIS OF SYNTHETIC MIXTURE (N = 5) ACTUAL CONC. (mg) CONC. FOUND (mg) % CV % PURITY FAM % IBU % CONCLUSION First order derivative method for simultaneous estimation of Famotidine and Ibuprofen in synthetic mixture was developed and validated. The method was found to be accurate and it is more sensitive to the smallest changes in the concentration. It has advantage that it eliminates the spectral interference from one of the two drugs while estimating the other drug by selecting zero crossing point in the derivative spectra of each drug at selected wavelength. The % assay results of 99.52% for FAM and 99.33% for IBU indicate that the developed method was successfully utilized for the estimation of FAM and IBU in their synthetic mixture. ACKNOWLEDGEMENT The authors are thankful to Vaibhav Analytical Laboratories, Ahmedabad, India for providing standard sample of drugs and also to the Arihant School of Pharmacy & BRI, Adalaj, Gandhinagar, India for providing facilities to carry out research work. IC Value

9 REFERENCES 1. (DB00927) accessed on 12/11/ accessed on 12/11/ (DB01050) accessed on 12/11/ accessed on 12/11/ The Indian pharmacopoeia: 4 th edn, Vol. 2, New Delhi, Controller of publication, 2010: & The United States pharmacopoeia: 28threvs rockville md, US Pharmacopoeial convention. Inc, 2009: 2344, 2345, 2607 and British pharmacopoeia: Vol. 2, London her majesty s stationary office, 2010: 860, Japanese pharmacopoeia: 15th edn, Society of Japanese pharmacopoeia, Tokyo 2006: 501, European Pharmacopoeia Vol 6.0.: European Directorate for the Quality of Medicines: 1865, 1866, 2119 and ICH, Q2 (R1). Validation of Analytical Procedures: Text and Methodology: Mehta SA, Umarkar AR, Chaple V and Thote LT: Development of UV spectrophotometric methods for simultaneous estimation of famotidine and diclofenac potassium in combined dosage form using simultaneous equation method.journal of pharmacy research 2011; 4: Apostu M, Nela B and Dorneanu V: UV spectrophotometric assay of famotidine in combination with picrolonic acid, picrolinate. International Jouranal of Pharmaceutical Science 2009; 2: 109, Riddhi G, Rajashree M and Savaliya P: Development and validation of spectrophotometric methods for simultaneous estimation of ibuprofen and paracetamol in soft gelatin capsule by simultaneous equation method. International journal of chemtech research, coden (usa) 2010; 2: Tapan G and Krishna T: Simultaneous equation method for simultaneous estimation of paracetamol and ibuprofen in combined dosage form.international journal of pharmaceutical & biological archives 2010; 3: IC Value

10 15. Sachan A and Trivedi P: First derivative spectrophometric estimation of ibuprofen and destropropoxyphene.hcl in solid dosage formulation. Royal society of chemistry journals 2009; 2: 220. For Correspondence: Deep P. Patel deep.jerry@gmail.com IC Value