Spectrophotometric Determination of Mefenamic Acid in Pharmaceutical Preparations Via Arsenazo III – Cerium ( III ) Reaction *

A sensitive indirect spectrophotometric method is proposed for determining mefenamic acid in pure form and in its pharmaceutical preparations. The method is based on oxidation-reduction reaction between mefenamic acid and cerium (IV) ion, and subsequent Ce(III) reaction with arsenazo (III) reagent in acidic medium to produce a greenish-blue complex which is stable, water soluble and has a maximum absorption at 654 nm with a molar absorptivity of 1.731×10 l.mol.cm. Beer’s law is obeyed in the concentration range from 1 to 10 μg mefenamic acid in a final volume of 25 ml. The proposed method has been applied successfully to determine mefenamic acid in pharmaceutical preparations. ــــــــــــــــــــــــــــــــــــــــــــــــــــــ تسملا يف كيمانيفيملا ضماحل يفيطلا ريدقتلا ح لعافت مادختساب هينلاديصلا تارض يثلاثلا مويريسلا عم يثلاثلا وزا نيسرلاا صخلملا ةيفيط ةقيرط حارتقا مت ةساسحو ةرشابم ريغ ريدقتل نيفيملا ضماـح ا كـيم لا هلكشـب يـقن يـفو رضحتسم هتا يئاودلا ه . تدمتعا ةقيرطلا ىلع لااو ةدسكلأا لعافت لازتخ نيب نيفيملا ضماح ا و كـيم نوـيأ مويريسلا يعابرلا لعافتلا جتان ةلعافم اهعبتي وزآ نيسرآ فشاكلا عم يثلاثلا مويريسلا نويأ III طسو يف دقعم يطعيل يضماح رضخا  ً ا ً ا قرزم نوللا يئاملا لولحملاب بئاذو رقتسم ُ ت اق دـنع هصاصـتما ةدش س يجوملا لوطلا 654 رتيمونان صاصتملاا ةميق تناكو ةي يرلاوملا ة 1.731 × 10 رتل . لوم 1 . مس 1 و نا قبط نيب زيكرتلا ىدم يف ريب نوناق 1 و 10 نم مارغوركيام يملا ضماح نيف ا كيم يئاهن مجح يف 25 لم . مـت ريدقت يف حاجنب ةحرتقملا ةقيرطلا قيبطت نيفيملا ضماح ا كيم يف رضحتسم هتا ةيئاودلا . ـــــــــــــــــــــــــــــــــــــــــــ ــــــ ـــــ * خيراتب دقعنملا ءايميكلل لولأا يملعلا رمتؤملاب هءاقلإ مت 22 23 ناسين 2008 ءايميكلا مسق ىف / مولعلا ةيلك / لصوملا ةعماج Nabeel S. Othman and Lena S.Awadis 9 INTRODUCTION Mefenamic acid [2-(2,3-dimethyl phenyl)amino]benzoic[I] acid is a non-steroidal anti-inflammatory drug which has analgesic, anti-inflammatory and antipyretic actions. It is used specially in the treatment of rheumatoid arthritis, osteoarthritis and other muscular-skeletal diseases (Martindale, 1982).


INTRODUCTION
Mefenamic acid [2-(2,3-dimethyl phenyl)amino]benzoic [I] acid is a non-steroidal anti-inflammatory drug which has analgesic, anti-inflammatory and antipyretic actions.It is used specially in the treatment of rheumatoid arthritis, osteoarthritis and other muscular-skeletal diseases (Martindale, 1982).

CooH
Me N Me H

[I] M.wt =241.3 g.mol¯1
The standard method for the assay of the pure drug is titrimetry, using sodium hydroxide as a titrant and phenol red as indicator (British pharmacopia, 2000).
Different techniques have been described for the determination of mefenamic acid: High performance liquid chromatography has been used for the determination of the drug in biological fluids such as human plasma (Niopas and Mamzoridi, 1994) or in human serum (Mohammed et al., 2004), and in horse plasma (Ishidaka et al., 1986).
Terbium sensitized fluorescence is used to develop a sensitive spectrofluorometric method for determination of mefenamic acid (Pinclopi et al., 1998), another spectrofluorimetric method based on oxidation of mefenamic acid with cerium (IV) (Tabrizi, 2006 ).
Many methods have been used in simultaneous determination of mefenamic acid in the presence of another drug such as paracetamol (Dinc et al., 2002 ), ethamsylate (Garg and Saraf, 2007).
However some of these procedures suffer from one or another disadvantage such as: the product may be extracted to organic solvent (Idowut et al., 2002), or require nonaqueous medium (El-Sherif et al., 1997) and other required control of temperature (Aman et al., 2005, Tabrizi, 2006).
Arsenazo (III) undergoes sensitive and selective reactions with several cations, such as reaction with cerium (III) ion in the presence of cerium (IV) ion.This reaction can be used in the determination of some organic compounds which have the ability to undergo COOH oxidation-reduction reaction with cerium (IV) ion (Al-Irhayim, 2004;Al-Abdaly, 2005;Jaime and William, 1991).
The objective of investigation reported in this paper is to develop a simple and accurate spectrophotometric method for the determination of mefenamic acid based on the oxidation of mefenamic acid with cerium (IV) ion, and the produced cerium (III) ion is subsequently reacted with arsenazo (III) reagent in acidic medium to form highly coloured complex.

EXPERIMENTAL
All spectrophotometric measurements are performed on Shimadzu UV-visible recording spectrophotometer UV-160 using 1-cm silica cells.pH meter type Philips PW 9420 is used for pH reading.

Reagents
All chemicals used are of analyticalreagent grade.
Standard mefenamic acid solution, 100µg.ml - .This solution is prepared by dissolving 0.01 g of pure mefenamic acid (SDI-Iraq) in a solution of sodium hydroxide (0.1N) containing 5 ml ethanol and the volume is diluted to 100 ml with sodium hydroxide (0.1N) in a volumetric flask.This solution was further diluted to 10µg.ml -1 with distilled water.Ammonium ceric sulphate [cerium(IV) ion solution], 6.6×10 -5 M.This solution is daily prepared by dissolving 0.0105 g of ammonium ceric sulphate dihydrate (BDH) in 250 ml of distilled water in a volumetric flask.
Arsenazo III reagent solution,2×10 -4 M.This solution is prepared by dissolving 0.0411 g of arsenazo III (Fluka) in 250 ml distilled water in a volumetric flask.
Hydrochloric acid solution, 0.05 N.This solution is prepared by appropriate dilution of 4.2 ml of the concentrated hydrochloric acid (11.8 N) solution to 100 ml with distilled water in a volumetric flask.
Buffer solution, pH 3.This solution is prepared by mixing 50 ml of 0.1N glycine solution (0.3752g glycine dissolved in 50ml distilled water) with 5.7ml of 0.2N HCl, then the volume is completed to 100 ml with distilled water and adjusted to pH 3 by pH meter.

Procedure and calibration graph
To a series of 25-ml calibrated flasks, an increasing volume (0.1-1.8 ml) of 10 µg.ml -1 mefenamic acid solution are transferred to cover the range of calibration graph, followed by 9 ml of 6.6 ×10 -5 M cerium (IV) ion solution and 0.9 ml of 0.05N HCl, the solutions are left for 25 minutes at room temperature, followed by addition of 2 ml of 2×10 -4 M arsenazo (III) reagent solution then 3 ml of pH 3 was added and diluted to the mark with distilled water, the absorbances are measured at 654 nm against the reagent blank.Beer's law is obeyed over the range of concentration 1 to 10 µg mefenamic acid in 25 ml (Fig. 1).A negative deviation from Beer's law is occurred beyond the upper determination limit.The apparent molar absorptivity, referred to mefenamic acid, has been found to be 1.731 ×10 5 l.mol -1 .cm - .Fig. 1:Calibration graph for mefenamic acid determination using the reaction between arsenazo(III) and cerium(III).

Determination of mefenamic acid in capsule.
Weight and mix the contents of five capsule (each one contains 250 mg mefenamic acid), an accurately weighed amount of powder equivalent to 0.01g mefenamic acid is dissolved in a mixture of 5 ml ethanol and 75 ml of 0.1N NaOH, after filtration the filtrate is completed to 100 ml with a 0.1N NaOH in a volumetric flask.A 10 ml of the above solution is diluted to 100 ml to prepare 10µg ml -1 .A suitable aliquot of solution was taken and the recommended procedure was followed for analysis of the drug.Determination of mefenamic acid in suspension.The content of the container (100 ml, each 5ml contains 50 mg mefenamic acid) is dissolved in a mixture containing 25 ml of ethanol and 100 ml of 0.1N NaOH then the solution is warmed, then filtered and the volume is completed to 250 ml with 0.1N NaOH, 2.5ml which is equivalent to 0.01 g mefenamic acid is transferred into a 100.ml calibrated flask and the volume is completed with a distilled water.A 10 ml of the above solution is diluted to 100 ml to prepare 10µg ml -1 solution.A suitable aliquot of solution was taken and the recommended procedure was followed for analysis of the drug.

RESULTS AND DISCUSSION Absorption spectra
Absorption spectrum of the coloured complex formed from the reaction between cerium (III) ion with arsenazo (III) in acidic medium against its corresponding reagent blank shows maximum absorption at 654 nm in contrast to the arsenazo reagent blank (Fig. 2).

Optimization of reaction conditions
The effect of various parameters on the absorption intensity of the coloured complex containing 10µg of mefenamic acid is studied and the reaction condition have been optimized.

Effect of pH
The effect of pH on the intensity of coloured complex is examined.Different volumes 0 -3 ml of 0.05M hydrochloric acid solution is added to an aliquot of solution containing 10 µg of mefenamic acid.The intensities of absorption were measured against the reagent blank at 654 nm.The results are shown in Table 1.The results shown in Table 1 indicate that the pH of 3.09 (0.9 ml of 0.05N HCl) is considered optimum.A pH 3 is selected for subsequent investigation because of good sensitivity.Five buffer solutions of pH 3 with different composition have been tested, tartaric acid-NaOH (B 1 ), citric acid-NaOH (B 2 ), KH phthalate-HCl (B 3 ), glycine-HCl (B 4 ) and formic acid-NaOH (B 5 ) (Table2).The results shown in Table 2 indicate that all types of buffer solutions decrease the intensity of the coloured complex, but the results improved when buffer solution added after adding 0.9 ml 0.05 N HCl.3ml of glycine-HCl buffer solution (B4) is recommended in the subsequent experiments according to the highest intensity of the complex and it produces more stable complex (Table 3).

Effect of oxidizing agent [cerium(IV) ion] amount
The effect of cerium (IV) ion concentration on the absorbance of the complex was studied.It was found that 9 ml of 6.6×10⎯ 5 M of Ce(IV) gave maximum absorption which is recommended in subsequent experiments (Table 4).

Effect of time on reduction of cerium (IV) ion
The effect of time needed to complete the reduction of cerium (IV) ions to cerium (III) ions was studied by standing of the solutions after adding cerium (IV) ion solution for different times at room temperature, then the other reagents were added and the absorbances were measured against the reagent blank (Table 5).The results indicate that complete reduction of cerium (IV) ions needed 25 minutes and the increase in intensity at 30 minutes and above is considered tolerable from that at 25 minutes.Therefore, the standing time 25 minutes was recommended for the subsequent experiments.

Effect of arsenazo III reagent amount
The effect of the amount of arsenazo III (AzIII) reagent on maximum formation of the coloured complex is investigated.The results are shown in Table 6.The results shown in Table 6 indicate that 2 ml of arsenazo (III) reagent solution give higher sensitivity and higher value of correlation coefficient (r), therefore it has been selected for subsequent experiments.

Effect of order of addition of reagents
The effect of order of addition of reagents on the absorbance of coloured complex is investigated (Table 7).The results indicate that the order of addition of reagents should be followed as given under procedure (order I).

Effect of Time
The effect of time on the development and stability of the coloured complex for different amounts of mefenamic acid was investigated under the optimum experimental conditions established.The colour formation occurs immediately after the addition of all reaction mixtures and the absorbance of the complex remains constant for at least 1 hour (Table 8).The above stability period is sufficient to allow several measurements to be performed sequentially.

Effect of interferences
The effect of some foreign compounds which often accompanied pharmaceutical preparations are studied by adding three different amounts (100, 500 and 1000µg) to 10 µg of mefenamic acid in a final volume 25 ml (Table 9).The results in Table 9 indicated that the studied foreign compounds did not interfere in the determination of mefenamic acid by using the proposed method except Arabic gum at high concentration.

Accuracy and precision
To check the accuracy and precision of the calibration graph, mefenamic acid was determined at three different concentrations.The results shown in Table 10 indicate that the calibration graph is satisfactory.

Nature of the reaction between mefenamic acid and cerium(IV) ion
Job's method was used in the determination of the reaction ratio of mefenamic acid with cerium (IV) ion.The results obtained (Fig. 3) showed that a 1:1 mefenamic acid to cerium (IV) ion ratio is obtained.The probable mechanism of the reaction might be the following:

Me
Me N

Nature of arsenazo (III)-cerium (III) ion complex
The stoichiometry of arsenazo III and cerium ion, the reaction is investigated using the Job's method under the optimized conditions.The results obtained (Fig. 4) showed that a 1:1 arsenazo (III) to cerium (III) ion ratio is obtained.

Analytical applications
The proposed method was applied to determine mefenamic acid in different pharmaceutical preparations.On applying proposed procedure, good recoveries were obtained as shown in Table 11.

Evaluation of the proposed method
The performance of the proposed method is assessed by calculating the student ' s ttest compared with the standard method (British Pharmacopoeia, 2000).At the 95% confidence limit for four degree of freedom, the calculated t -values do not exceed the theoretical value (2.776).The results in Table 12 indicate that there is no significant difference between the proposed method and the standard method.Standard addition method has been used in determination two of drugs under investigation in order to prove that the proposed method is applied to the determination of mefenamic acid without interferences (Table 13 and Fig. 5).The results in Table 13 and Fig. 5 indicated that the proposed method can be applied successfully to the determination of mefenamic acid in pharmaceutical formulations .

COMPARISON OF THE METHODS
Table 14 shows the comparison between some of analytical parameters of the present method with another recent spectrophotometric method.The results in Table 14 show that the suggested method for the determination of mefenamic acid was sensitive and needs neither temperature control nor extraction step.

CONCLUSION
The proposed method for the determination of mefenamic acid in pharmaceutical preparations was sensitive.The complex formed was fairly soluble in aqueous solution.The statistical analysis of the results indicates that the method has good accuracy (average relative error between -0.92 to +1.65 %) and good precision (average relative standard deviation not more than 1.93 %).The t-value indicates that there was no significant difference between the proposed method and the standard method.Moreover, the proposed method needs neither temperature control nor extraction step.

Fig. 2 :
Fig.2:Absorption spectra of 10 µg mefenamic acid / 25 ml treated according to the recommended procedure and measured against (A) blank, (B) distilled water and (C) blank measured against distilled water.

Table 1 :
Effect of pH on absorbance

Table 2 :
Effect of buffer solution without and with hydrochloric acid on absorbance

Table 3 :
The stability of complex without and with buffer solution *Using hydrochloric acid only.** Using HCl + 3 ml of glycine-HCl

Table 4 :
The effect of ceric ion amount on absorbance

Table 5 :
Effect of time on reduction process

Table 6 :
Effect of arsenazo III reagent amount on absorbance

Table 7 :
The order of addition of reagents

Table 8 :
Effect of time on the absorbance of complex

Table 9 :
Effect of foreign compounds for assay of mefenamic acid

Table 10 :
Accuracy and precision

Table 12 :
Analysis of mefenamic acid in pharmaceuticals by proposed and official method *Average of five determinations

Table 13 :
The results of standard addition method *Average of three determinations