Spectrophotometric Determination of Mefenamic Acid via Oxidative Coupling Reaction with 4-Amminoantipyrine in Presence of N-Chlorosuccinimide

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1 ISSN X Pak. J. Anal. Environ. Chem. Vol. 9, No. 2 (2008) Spectrophotometric Determination of Mefenamic Acid via Oxidative Coupling Reaction with 4-Amminoantipyrine in Presence of N-Chlorosuccinimide Nabeel Sabeh Othman and Lena Sameer Awades Chemistry Department, College of Science, University of Mosul, Iraq Abstract A simple accurate and precise spectrophotometric method was proposed for determining mefenamic acid in pure form as well as dosage forms. The procedure is based on the oxidative coupling reaction of mefenamic acid with 4-aminoantipyrine reagent in an acidic medium (ph 3.6) in the presence of N-chlorosuccinimide as oxidizing agent to produce an intense colored and water-soluble product which exhibits maximum absorption at 588 nm. Beer s law is obeyed in the concentration range of g mefenamic acid in a final volume of 25 ml ( ppm). The proposed method was applied to determine mefenamic acid in pharmaceutical preparations. The amount of mefenamic acid found are very similar to those obtained by a standard method. Keywords: Mefenamic acid, Oxidative coupling, 4-Amminoantipyrine, N-chlorosuccinimide Introduction Mefenamic acid [2-(2,3-dimethyl phenyl)amino] benzoic acid is a non-steroidal drug which has analgesic, anti-inflammatory and antipyretic actions and it is used specially in the treatment of rheumatoid arthritis and osteoarthritis and other muscular-skeletal diseases []. Various methods have been reported for the determination of mefenamic acid as pure and in dosages forms. These methods include titrimetric [2,3], chromatographic [4-6], luminescence [7] flow injection [8, 9], electrometric [10], spectroflouro- metric [11,12] and spectrophotometric methods [13-19], Also spectrophotometric methods have been described for the simultaneous determination of mefenamic acid in the mixture with other active drugs in the same pharmaceutical preparation [20-21]. However, some of these procedures suffer from one or another disadvantage such as extraction into organic solvent [13], requiring non-aqueous medium [17] and others need control of temperature [16,18]. In the present work a new, simple and precise method is proposed for the determination of *Corresponding Author abdelrahmanbasil@yahoo.com mefenamic acid in pharmaceutical preparations. The method is based on oxidative coupling reaction of mefenamic acid with 4-amminoantipyrine in presence of N-chloro-succinimide and the absorbance of the colored product measured at 588 nm. Experimental Apparatus All spectrophotometric measurements were performed on Shimadzu UV-visible recording spectrophotometer UV-160 using 1-cm silica cells. ph meter type Philips PW 9420 was used for ph reading.. Reagents All chemicals used are of highest purity available. A 100 g.ml -1 solution of mefenamic acid (obtained from state drug industry, Samara, Iraq) was prepared by dissolving appropriate amount of mefenamic acid in 100 ml ethanol and was kept in refrigerator. The N-chlorosuccinimide solution of concentration M and M of 4- aminoantipyrine solution were also prepared by

2 65 Pak. J. Anal. Environ. Chem. Vol. 9, No. 2 (2008) dissolving accurate weight in distilled water. The buffer solution (ph 3.6) was prepared by mixing 50 ml glycine (0.1M) with 2.5 ml HCl (0.2M) then the solution was diluted with distilled water to the mark in 100 ml calibrated flask.. General procedure and calibration graph In 25 ml volumetric flasks, 3 ml of glycine buffer (ph 3.6) and 7 ml ( M) of 4- aminoantipyrine were mixed, then ml of mefenamic acid (100 g ml -1 ) to cover the concentration rang of g mefenamic acid / 25 ml ( ppm), and 3 ml of N-chlorosuccinimide ( M) were added then the flasks were allowed to stand for 5 minutes. After the volumes were completed to the mark with distilled water, the absorbance of colored product was measured at 588 nm against the reagent blank, a linear calibration graph was obtained over the concentration range of g mefenamic acid / 25 ml ( ppm), a concentration above 250 g mefenamic acid (M.A) / 25 ml gave a negative deviation from Beer s law (Fig. 1). The molar absorptivity had been found to be l.mol -1.cm -1. Absorbance y = x r = µg of M. A / 25ml Figure 1. Calibration graph for determination of mefenamic acid. Procedure for the M.A capsule The contents of 10 capsules of M.A (State drug industry, Samara, Iraq, 250 mg/capsule) were finely ground. An accurately weighed powdered sample containing 0.01g of MA was dissolved in 100 ml ethanol to prepared 100 g ml -1 of M.A. Different volumes of this solution were transferred and then, proceed as described under general procedure and calibration graph. Procedure for the M.A suspension A 100 ml suspension (State drug industry, Samara, Iraq, 50 mgm.a/5 ml) was mixed with 115 ml ethanol then the solution was diluted to 250 ml (after filtration) with ethanol in a calibrated flask, 2.5 ml of above solution was diluted to 100 ml with ethanol to prepare a solution of 100 g MA. Different volumes of this solution were transferred and then, proceed as described under general procedure and calibration graph. Results and Discussion The effect of various parameters on the intensity of the colored product (from 100 g M.A/25ml was used) was studied and the reaction conditions were optimized. Different types of available oxidizing agents were used to select the best one which gave the highest intensity (Table1). Table 1. Selection of oxidizing agent. Oxidizing agent* ( M) Absorbance ** N-Chlorosuccinimide N-bromosuccinimide Potassium periodate Potassium chromate Potassium dichromate Ammonium ceric Sulphate Turbid *3 ml of oxidizing agent is used. ** = maxs - maxb Where S = The colored product, B = Blank The results illustrated in table 1 indicated that N- chlorosuccinimide (NCS) gave the highest intensity of colored product and a good color contrast. Another experimental results indicated that a 3 ml of NCS ( M) gave high intensity of the colored product and therefore it was recommended for the subsequent experiments. The effect of the time needed to complete the oxidative coupling reaction had been studied. The results indicated that maximum intensity of the colored product at 588 nm occurred after 5 minutes before dilution of the flask with distilled water, then a slight decrease in intensity with increasing time, so that it was recommended for the subsequent experiments (Table 2). Table 2. The effect of time on oxidizing coupling reaction. Time(min.) Absorbance

3 Pak. J. Anal. Environ. Chem. Vol. 9, No. 2 (2008) 66 The amount of 4-amminoantipyrine (4-AAP) reagent affected the intensity of the colored product, the absorbance of the colored product attained maximum intensity on using a volume of 7 ml of M 4-AAP. The effect of ph on the intensity of the colored product was studied using solution for ph reagents from , it was found that the optimum ph was equal to Therefore different buffer solution of ph value equal to 3.6 were prepared (0.2M acetic acid + 0.2M sodium acetate; B 1 ), (0.1M glycine + 0.2M HCl; B 2 ) and (0.1M citric acid + 0.1M sodium citrate; B 3 ) and added from 3 to 5 ml with different orders of addition. The results indicate that 3 ml of B 2 added after M.A gives the highest intensity of the colored product (Table 3). Table 3. The effect of buffer. Absorbance/ ml buffer solution added Order of B1 B2 B3 addition 3 ml 5 ml 3 ml 5 ml 3 ml 5 ml *I II III *I-M.A (S) +Buffer(B)+NCS+ 4-aminoantipyrine( 4-AAP) II - S+NCS+B+R III- S+NCS+R+B Absorbance without buffer =0.372 Different orders of addition of the reagents had been done. The results indicate that the order in the recommended procedure gave the highest intensity and was selected in the subsequent experiments. The stability of the complex was estimated by following the color development at room temperature (25 1 C) and on thermostatically controlled water bath at zero and 45 C. It was observed that the sensitivity reached maximum at 25 C with good stability of complex with time for 60 minutes (Table 4), this stability period was sufficient for several measurements to be performed at the same time,so that the room temperature (25 1 C) was selected in the subsequent experiments. Table 4. The accuracy and precision. Amount of M.A. taken ìg Relative error, %* Relative standard deviation, %* Final absorption spectrum When M.A was treated according to the recommended procedure, the absorption spectrum showed a maximum absorption at 588 nm versus the blank (Fig.2). Figure 2. Absorption spectrum of (A)the colored product against blank, (B) complex against distilled water (C) blank against distilled water. To check the accuracy and precision of the calibration graph M.A was determined at three different concentrations and the result were shown in Table5 which indicated good accuracy and precision. Table 5. The accuracy and precision. Amount of M.A. taken µg Relative error, %* Relative standard deviation, %* *Average of five determinations Interferences The effect of the presence of some common excipients (glucose, lactose, starch, Gum Arabic and glycerin) on the selectivity of suggested method has been studied. The results indicate that there was no significant interference produced by these foreign substances (10-fold amount added) on the suggested method. Stoichiometry of mefenamic acid - 4-AAP complex The composition of the formed complex had been established using Job's method [22] the method was based on the measurement of series of solution in which molar concentration of mefenamic acid and 4-AAP vary but their sum remained constant, the other reagents added as mention in the general procedure and

4 67 Pak. J. Anal. Environ. Chem. Vol. 9, No. 2 (2008) calibration graph. The results indicate that the complex had been formed in a ratio 1 (M.A): 2(4-AAP). The para positions to the amino group was favored for coupling on both rings [13]. The probable reaction path might be written as follow : Table 7. The comparison of the methods. Analytical parameters Present method Literature method() Literature method() max (nm) Temperature ( C) Room temperature Using boiling water bath 30 ϩ C + Medium of reaction Reagent Beer s law range (ppm) Molar absorptivity (l.mol -1.cm - 1 ) Aqueous 4-Aminoantipyrine Absolute ethanol p-dimethyl aminocinnamaldehyde Non-aqueous 4-Amino-3,5- dinitrobenzoic acid RSD (%) Analytical applications The proposed method was successfully applied to determine M.A in its pharmaceutical preparations, capsule and syrup. The results are shown in Table 6. The results indicate that the M.A content measured by the present method was in excellent agreement with those obtained by the manual reference British pharmacopoeia method [2]. A comparison using t-test at confidence level (95%) and for four degrees of freedom indicate that there is not significant difference among the achieved results using these two methods. Table 6. Analytical application of proposed method. Pharmaceutical preparation g mefenamic Acid present /25ml g mefenamic Acid measured /25ml Recovery*, (%) Ponstidin capsule (250mg) N.D.I-Iraq Ponstidin capsule (250mg) GMBH,Germany Mefastan(50mg/5ml) AL-Mansour Pharma- Ind.(Baghdad-Iraq) *Average of three determinations A comparison of the present method with some of existing spectrophotometric methods was given in Table 7 which demonstrates the advantages of proposed method. Conclusion A new simple and sensitive spectrophotometric method for the determination of trace amounts of mefenamic acid in aqueous solution based on reaction of mefenamic acid with 4-AAP in the presence of NCS was concluded. The proposed method has been successfully applied to assay of mefenamic acid in various pharmaceutical preparations.. References 1. Martindale, The Extra Pharmacopoeia, 28th Edn. The Pharmaceuticalpress,London., (1982) British Pharmacopoeia on CD-ROM, 3rd Edn., System Simulation Ltd, the stationary office, London, (2000). 3. O. Cakirer, E. Kilice, O. Atakol and A. Kener, J. Pharm. Biomed. Anal.,90 (1999) I. Niopas and K. Mamzoridi, J. Chromatogr. B Biomed. Sci. and Appl., (1994) R Mohammd., A. Ali, H. Yalda and A. Fakhredin, J. Chromatogr. B, 800 (2004) O. Ishidaka, T. Shinohara, T. Tanaka and A. Momose, Japan Analyst, 35 (1986) N. Arnaud and J. Georges, Anal. Chim. Acta., 476 (2003) M. Albero, C. Sanchez and M. Garcia, J. Pharm. Biomed. Anal., 13 (1995) A. Fatma, A. Salma, and A. Abdulrahman, Anal. Chim. Acta., 416 (2000) L. Liu and J. Song., Anal. Biochem., 354 (

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