Development and Validation of Ratio Spectra Derivative Spectrophotometric Method for Determination of Mefenamic acid and Ethamsylate in combined Formulation


Vinit D. Patil, Ajay S. Bhadoriya, Kunal D. Ingale, Aniruddh R. Chabukswar,  Vishnu P. Choudhari* and Bhanudas S. Kuchekar

Department of Pharmaceutical Analysis  and Quality Assurance, MAEER’s Maharashtra Institute of Pharmacy, Kothrud, Pune-411038, India.

*Corresponding Author E-mail:



A simple, economical, precise and accurate ratio spectra derivative spectrophotometric method for Mefenamic acid (MEF) and Ethamsylate (ETH) combination has been developed.  The method is based on obtaining first derivative of ratio spectra and measuring amplitude at selected wavelength. The derivative amplitudes at 250.99 nm and 287.11 nm were selected for measuring the response of Mefenamic acid (MEF) and Ethamsylate (ETH) respectively. Beer’s law is obeyed in the concentration range of 10-30 μg mL-1 for both the drugs. The % assay was found to be in the range 98.0 – 101.0 % for MEF and 98.91 – 101.72 % for ETH by the proposed method. The method was validated with respect to linearity, precision and accuracy. Recovery was found in the range of 98.72 - 100.64 % for MEF and 98.45 – 101.21 % for ETH.


KEYWORDS: Mefenamic acid, Ethamsylate, Ratio Spectra, First Derivative.



The resolution of complex multicomponent systems without separation of the constituting analytes is rather a difficult task. The resolution of binary mixtures of compounds with overlapped spectra by derivative technique is frequently made on the basis of zero-crossing measurements.1–3


Ethamsylate (ETH); 2,5-Dihydrobenzene sulphonic acid     ( Fig. 1) is haemostatic drug. It reduces capillary bleeding when platelets are adequate; exerts antihyaluronidase action-improves capillary wall stability but does not stabilize fibrin (not an antifibrinolytic). It inhibits PGI2 synthesis and hence promotes platelet aggregation. It has been used in the prevention and treatment of capillary bleeding in menorrhagia, after abortion, PPH, epistaxis, malena, hematuria and after tooth extraction.


Mefenamic acid (MEF); N-(2, 3-Xylyl)-2-aminobenzoic acid is analgesic, antipyretic and weaker non-steroidal anti-inflammatory drug used as combination drug therapy for treatment of painful menstruation. It inhibits COX and antagonises certain actions of PGs. It inhibits leukotriene level as well, by inhibiting phospholipase A2.


It exerts peripheral as well as central analgesic action. Recently, the combination of MEF and ETH has demonstrated significant activity against painful menstruation.


For the individual determination of MEF and ETH spectrophotometric methods are available such as Area under Curve, Dual Wavelength method4,5, HPLC method for the simultaneous estimation of the MEF and ETH in tablets6, and a spectrophotometric Simultaneous equation method  is reported  for this combination7.  To our knowledge Ratio Spectra Derivative Spectrophotometric Method for the combination is not available. By ratio derivative method analytes can be estimated even in presence of interfering species therefore it was felt necessary to develop and validate simple, rapid, selective and low cost Ratio Spectra Derivative Spectrophotometric Method8 for the simultaneous determination of MEF and ETH as per ICH guidelines.



2.1. Instruments:

The spectrophotometric measurements were carried out on an UV-Visible double beam spectrophotometer (Varian Cary -100) with 10 mm matched quartz cells were used. All weighing Shimadzu AUW-220D balance was used.


Figure1. Chemical structures of drugs


2.2. Materials and Reagents:

Spectroscopy grade methanol was used throughout the study. Pure drug sample of MEF and ETH were kindly supplied as a gift sample by Emcure Pharmaceuticals Pvt. Ltd. Pune, India. These were used without further purification. Tablets were purchased from local market; containing MEF 500 mg and ETH 500 mg. Tablet used for analysis were of brand Sylate-M from two different batches {Batch No. ECA09001 (Formulation-I) and PDB09001 (formulation-II)} containing 500 mg of each drug, manufactured by Emcure Pharmaceuticals Pvt. Ltd. Pune, India.


2.3. Preparation of standard solutions and Construction of calibration graphs:

Stock solutions containing 1 mg mL-1 of MEF and ETH were prepared separately in methanol. Stock solutions were diluted suitably in methanol. Solutions containing MEF and ETH in combination in concentration range of 10 to 30 µg mL-1  were prepared and scanned. Solutions of MEF and ETH, 20 µg mL-1 of each were prepared separately in methanol and scanned and these scans were used to construct calibration curve for both the drugs.


2.4. Preparation of Sample Solution and Formulation Analysis:

Twenty tablets were weighed accurately and a quantity of tablet powder equivalent to 100 mg of MEF  (100 mg of ETH) was weighed and dissolved in the 80 mL of methanol with ultrasonication for 5 min and solution was filtered through Whatman paper No. 41 into a 100 mL  volumetric  flask. Filter paper was washed with methanol, adding  washings to the volumetric flask and volume was made up to the mark with methanol. The solution was suitably diluted further to get required final concentration 20 µg mL-1 of both the drugs. The solutions were scanned within the wavelength range 200–400 nm and scan was stored.



3.1. Ratio spectra first derivative spectrophotometry:

The absorption spectrum of mixture is divided by the absorption spectrum of std. solution of one of compound and first derivative of ratio spectrum is obtained, resulting spectra is independent of conc. of divisor. The conc. of active compounds are then determined from calibration graph obtained by measuring amplitude at points corresponding to minima or maxima. Using appropriate dilutions of standard stock solution, the two solutions were scanned separately (Fig 2). The ratio spectra of different MEF standards at increasing concentrations were obtained by dividing each with the stored spectrum of the standard solution of ETH (20 μg mL-1) as shown in Fig 3 and the first derivative of these spectra traced, are illustrated in Fig 4. Wavelength 250.99 nm was selected for the quantification of MEF in MEF + ETH mixture. Likewise, the ratio and ratio derivative spectra of the solutions of ETH at different concentrations were obtained by dividing each with the stored standard spectrum of the MEF (20 μg mL-1) (Fig. 5). Wavelength 287.11 nm was selected for the quantification of ETH in MEF + ETH mixture. Measured analytical signals at these wavelengths were proportional to the concentrations of the drugs. The amount of MEF and ETH in tablets was calculated by using following equations9


At 250.99 nm: C MEF =(Ratio derivative amplitude for MEF - 0.0117) / 1.056 .... (1)

At 287.11 nm: C ETH = (Ratio derivative amplitude for MEF - 0.0608) / 0.098 ..... (2)


Figure2. Zero order spectra of Mefenamic acid, Ethamsylate and mixture in methanol at shown concentration.


Figure 3 Ratio spectra of Ethamsylate (conc. 10 - 30 μg mL-1)


Figure 4. First derivative ratio spectra of Mefenamic acid (conc. 10 - 30 μg mL-1)    


Figure 5. First derivative ratio spectra of Ethamsylate(conc. 10 - 30 μg mL-1)


3.2. Method validation:

Method was initially applied to mixtures of standard drug solutions, after getting satisfactory results it was then applied to tablet formulation.


In order to test the accuracy of the proposed method, synthetic mixtures of each combination were prepared in different proportions. The resulting mixtures were assayed according to the above stated procedure and the results were calculated as the percentage of analyte recovered. The good recovery values and the low values of the standard deviation assure the high accuracy of the proposed method


The linearity of the proposed method was evaluated for each drug by analyzing different concentrations of each of MEF and ETH, within the concentration range in Table 1 in the absence and presence of certain concentrations of other components in the mixture. The assay was performed according to the previously stated conditions. The corresponding first derivative amplitude for each drug was measured at the specific wavelength, and plotted against its concentration (Table 1). A straight line was obtained in each case. Statistical analysis of these graphs showed excellent linearity of the calibration graph and agreement to Beer’s law. Moreover, the slope was independent of the concentration of each component in the mixture.


Table -1 Regression equation and results of assay, precision and recovery





Wavelength (nm)



Range (μg mL-1)



Tablet Assay

( % RSD)

Batch I

100.02, 0.35

98.97, 0.67

Batch II

99.95 , 1.2





( % RSD)







99.88, 0.56

100.06, 0.53



99.98, 0.76





( %RSD)

Repetition (n=6)



Intra Day (n=3x3)



Inter Day(n=3x3)












Correlation coefficient  (r)




















Statistical data in the result shows precision, recovery and %RSD was always less than 2% indicate precision and accuracy of the method.


Method selectivity was detected by preparing different mixtures within the linearity range such that the mixture contains variable amounts of one component and constant amounts of the other component. The mixtures were then analyzed according to the above stated procedures and the first derivative ratios were obtained. Statistical analysis of these data showed that the slope of the calibration graph for each drug is independent on the concentration of the other components (Table 1). It only shows that the first derivative amplitudes were only a function of the concentration of the assigned drug at the specified wavelength. Consequently, the results obtained verified the high selectivity of the proposed method and its potential for the simultaneous determination of this mixture.



The proposed method is simple, accurate, and precise  for quantitative analysis of MEF and ETH as a binary mixture. The proposed method is simple as there is no need for solvent extraction and it estimates each component independent of the other. In addition, the method is rapid, of low cost, and harmless to the environment. Therefore it could be applied in quality control laboratories where economy and time are essential.



The authors wish to express their gratitude to Emcure Pharmaceuticals Pvt. Ltd. Pune, India, for the sample of pure MEF and ETH. The authors are also thankful to the management of MAEER’s Maharashtra Institute of Pharmacy for providing necessary facilities.



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Received on 11.03.2010       Modified on 30.03.2010

Accepted on 07.04.2010      © RJPT All right reserved

Research J. Pharm. and Tech.3 (3): July-Sept. 2010; Page 921-923