Indirect Spectrophotometric Method for the Determination of Mefenamic Acid in Pharmaceutical Formulations *

A simple, sensitive and accurate spectrophotometric method for the determination of mefenamic acid in pure form and in some pharmaceutical formulations was developed. The method is based on the oxidation of mefenamic acid by iron(III), and subsequent complexation of iron(II) with o-phenanthroline, forming a red-colored complex (ferroin) having the maximum absorbance at 510 nm. Beer's law is obeyed in the concentration range of 0.4-2.0 μg\ml, the molar absorptivity and Sandell's sensitivity are 2.9×10 L.mol.cm and 8.3 ng.cm, respectively and the relative standard deviation (RSD) is less than 2.0 (n=10). The limits of detection and quantitation are 0.065 and 0.195 μg.ml,respectively. The method is applied successfully to determination of mefenamic acid in some pharmaceutical formulations (capsules and tablets). The common excipients do not interfere with the proposed method. A statistical comparison of these results with those of official method using (t and F) values at 95% confidence level shows good agreement and indicates no significant difference in the precision and the present method has good validity. Keyword: Mefenamic acid, Spectrophotometry; Iron (ш); phenanthroline ـ ـــــــــــــــــــــــــــــــــــــــــــــــــ ــ ريدقتل ةرشابم ريغ ةيفيط ةقيرط ضماح اح يف كيمانيفيملا ل تارضحتسم ضعب نمو ةيقنلا هت ه

Mefenamic acid is used in musculoskeletal and joint disorder such as rheumatoid arthritis, osteoarthritis (Martindale, 1999) and primary dysmenorrheal (Zhang andLi, 1998 andRemington,1995).Different methods for the determination of mefenamic acid have been described, such as titrimetry (official method) for the assay of pure form and pharmaceutical preparation using sodium hydroxide as titrant and phenol red as indicator (British pharmacopeia, 2005), HPLC (Chao et al., 1997 andHirai et al., 2002) gas chromatography (Chao, 1997) and cloud point extraction with spectrofluorimetry and spectrophotometry.(Tabrizi, 2006).
The most widely used methods for the determination of mefenamic acid are spectrophotometric methods (Idown et al., 2002;Sastry and Rao,1989;Sastry andRao, 1987, Dinc et al., 2002;Gangwal et al.,1996;Sastry and Rao,1989;Aman et al., 2005, Zommer et al.,1986and Alarfaj et al., 2009).However, all of these methods suffer from one or more disadvantage such as insufficient sensitivity, selectivity, tedious and use of complex solvent extraction procedures.Therefore, a simple method for assay of mefenamic acid is necessary for routine analysis and quality evaluation.

EXPERIMENTAL Apparatus
Spectro-scan 50 UV-visible (double beam) spectrophotometer with 1.0 cm quartz cells was used for absorption measurements, and Jenway 3310 pH meter was used for pH measurements.

Reagents
All chemicals used were of analytical or pharmaceutical grade and distilled water was used throughout.

Mefenamic acid stock solution:( 100 ppm).
This solution was prepared by dissolving 0.01 g of mefenamic acid in 100 ml 0.01 N NaOH solution.Mefenamic acid standard solution (10ppm) (4.1× 10 -5 M) This solution was prepared by diluting 10 ml of stock solution to 100 ml with distilled water in a volumetric flask.

Ferric chloride solution 0.1% in 0.01 N HCl
This solution was prepared by dissolving 0.1 g of ferric chloride in 100 ml of distilled water containing 2 ml of concentrated HCl

Buffer solution(pH 3)
This solution is prepared by mixing 16.2 ml of 0.1 M chloroacetic acid with 8.8 ml of 0.1M KOH, then the volume is completed to 100 ml with distilled water in a volumetric flask (Zommer and Bojarowicz,1986).

Recommended procedure
A known volume of sample solution containing 10-50 µg of mefenamic acid was transferred into a 25-ml calibrated flask followed by 3 ml of ferric chloride solution then 2 ml of 1,10-phenanthroline solution and 2 ml of buffer solution pH 3.0 were added, diluting the solution to the mark with distilled water, the flask was placed in a water bath maintained at 70 ±3 0 C for 15 min.The absorbance of the red-colored product was measured at 510 nm against a reagent blank.

Tablets
Weigh and powder 10 tablets.Dissolve a quantity of the powdered tablets containing 0.01 g of mefenamic acid in about 100 ml of 0.01 N sodium hydroxide.It was shaken thoroughly for about 10-15 min, and filtered.The filtrate was made up to 1 L with distilled water.Treate 3 ml of this solution as mentioned under recommended procedure.

Capsules
Dissolve a quantity of the mixed contents of 10 capsules containing 0.01 g of mefenamic acid in 100 ml of 0.01 N sodium hydroxide and mixed for 10-15 mint and then filtered.The filtrate was made up to 1L with distilled water.Treate 3 ml of this solution as described under recommended procedure.

RESULTS AND DISCUSSION
The method is based on the oxidation of mefenamic acid by iron (III) in the presence of o-phenanthroline in acidic medium.The iron (II) formed was quantitively and rapidly converted to the stable tris (o-phenanthroline) iron (II) complex (ferroin) having an absorption maximum at 510 nm (Marczenko, 1976)as shown below (Fig. 1) thus permitting the indirect estimation of mefenamic acid.The reaction variables were optimized by varying each variable while keeping others constant for obtaining maximum absorbance.The oxidation reaction was found to be quantitative in acidic medium, the pH 2.0-4.0 is considered optimum.Then, a pH of 3.0 is selected for the subsequent investigation.Four types of buffer solution pH3 of different compositions (Perrin and Dempsey, 1974) have been tested for this purpose.It was found that 1-3 ml of buffer B1 gives high sensitivity and 2.0 ml has been used for subsequent experiments.
The effect of the amount of FeCl 3 and o-phenanthroline amounts on the absorbance was investigated.A maximum and constant absorbance was found with 3.0 ml FeCl 3 and 2.0 ml of 1% o-phenanthroline, which were therefore adopted as being optimal.The color reaction occurred even at room temperature (25 0 C), though at higher temperature the color developed more rapidly.The maximum absorbance was observed after 15 min of heating at 70 0 C. A temperature of 70 0 C and a reaction time of 15 min were selected for reproducible results.Under the experimental conditions described, Beer's law is obeyed over the concentration range 0.4-2.0µg/ml Fig [2].Linear regression equation: A=0.0021+0.048C(r = 0.9993 n = 8).Where A is the absorbance and C is the concentration in µg\ml The apparent molar absorptivity was 2.9×10 4 L.mol -1 .cm - and Sandell's sensitivity was 8.3 ng.cm -2 .The limit of detection and quantification were evaluated as (Bassavaiah and Somashekar, 2007) Where b is the slope and So is the standard deviation of the regression line.The limit of detection was 0.065 µg.ml -1 and the limit of quantification as the lowest standard concentration which could be determined with acceptable accuracy, and precision was 0.195 µg.ml -1

ccuracy and precision
The accuracy and precision of the method were established by analyzing the pure drug solution at three different levels.The average recovery which is a measure of accuracy is 100± 0.92% revealing high accuracy of the method.The relative standard deviation (RSD), which is an indicator of precision, is better than ± 2.0% the results are complied in Table (2).

INTERFERENCES
To study the potential interference from the commonly used excipients and other additives such as povidone, starch, talc, microcrystalline cellulose, magnesium stearate and mannitol, recovery studies were carried out.Under the experimental conditions employed, to a known amount of drug (30 µg\25 ml), excipients in different concentrations were added and analyzed.Results of the recovery analysis are presented in Table (3).Excipients at the concentration shown in Table (3) don't interfere with the assay.In addition, recoveries in most cases were around 100 %.

APPLICATION OF THE PROPOSED METHOD
The proposed method was successfully applied to the analysis of mefenamic acid in capsules and tablets.The results of analysis for pharmaceutical formulations Table (4) were compared statistically by student t-test and by the variance ratio F-test with those obtained by official method at 95% confidence level.The calculated t -and F-values did not exceed the theoretical values indicating that there was no significant difference between the precision of the proposed and official methods.
Fig 1: Absorption spectra of A :30 µg/25 ml of mefenamic acid with FeCl 3phenanthroline against reagent blank.B:blank against distilled water.

Table 1 :
Table (1) shows the results of this investigation.Effect of different buffers on the absorbance

Table 2 :
Optical characteristics and statistical data for regression equation of the proposed method

Table 3 :
Determination of mefenamic acid in presence of excipients