INTRODUCTION:

Mefenamic acid is a nonsteroidal anti-inflammatory drug (NSAID) that exhibits anti-inflammatory, analgesic, and antipyretic activities in animal models. Chemically it is 2-[(2, 3-dimethylphenyl) amino] benzoic acid. The clinical application includes headache, dental pain, post-operative and post-partum pain and dysmenorrhoeae, in musculoskeletal and joint disorders such as osteoarthritis. It is official in Indian pharmacopoeia, British Pharmacopoeia and United State pharmacopoeia. Several analytical techniques like titrimetric, potentiometric, capillary isotachophoretic, spectrophotometric and HPLC have been reported for assay of mefenamic acid in pharmaceutical preparation and in human serum. However some of these methods are expensive and time consuming.

The main purpose of this investigation is to develop and validate HPLC method which is simple, rapid, precise and sensitive for estimation of mefenamic acid from pharmaceutical formulation.

EXPERIMENTAL^6-11:

MATERIALS:

Mefenamic acid pure drug was obtained as a gift sample from Ajanta Pharma Ltd., Mumbai and Diclofenac sodium pure drug was obtained from Bombay Drug House, Mumbai.

Methanol (HPLC), Acetonitrile (HPLC) were procured from Merck Laboratories, Mumbai. All the reagent solutions were prepared using double distilled water.

INSTRUMENT USED:

The HPLC system (JASCO), with PU 2080 plus intelligent HPLC pump, 20 µl sample loop injector (≠7725, Rheodyne, USA), UV-2075 plus intelligent UV/VIS detector with Borwin software version 1.5, LC-Net II/ADC system was used.

METHODS:

Preparation of mobile phase:

1000 ml of mobile phase was prepared by mixing 800 ml of acetonitrile and 200 ml of water, then pH was adjusted to 3.0 by addition of glacial acetic acid. The mixture was sonicated for 15 min. and filtered the resultant mixture through 0.45 µ nylon membrane filter.

Standard solution:^12-13

An accurately weighed quantity of about 25 mg of mefenamic acid was taken in 100 ml volumetric flask dissolved in sufficient quantity of water: methanol (60:40) and this was sonicated for 15 min and diluted to 100 ml with the same solvent so as to get the concentration of 250 µg/ml.

Determination of λ _max:

The standard solution of mefenamic acid was scanned at different concentrations in the range of 200-400 nm and the λ _max was determined.

Chromatographic condition:

The separation was performed on HiQ Sil C-8 (4.6*250 mm, i.d. 4.6 µm) column. The mobile phase was Acetonitrile:Water (80:20), pH adjusted to 3.0 with glacial acetic acid. The wavelength selected was 288 nm. The flow rate was 1.5 ml/min. The internal standard used was Diclofenac sodium.

Linearity and calibration curve:^14-17

The stock solution of mefenamic acid was diluted suitably to obtain calibration standards of 10-90 µg/ml. These solutions were injected (20µl) in triplicate and peak area was noted. Calibration curve for mefenamic acid was then plotted between peak areas against concentration at 288 nm.

Analysis of tablet formulation:^14-17

Twenty tablets, each containing 250 mg mefenamic acid were weighed and crushed to fine powder and quantity of powder equivalent to 25 mg mefenamic acid was weighed and transferred to 100 ml volumetric flask. Sufficient amount of mobile phase was added to dissolve the content and shaken for 15 min. The volume was made up to 100 ml with mobile phase and filtered through 0.45 µ nylon membrane filter. The filtered solution was injected and peak area was noted.

Method validation:^18-21

The method was validated for accuracy and recovery, precision, detection limit, quantitation limit, specificity and robustness according to ICH guidelines.

Accuracy:

The accuracy of the method was determined by calculating the recovery of mefenamic acid using standard addition method. For this purpose, a known amount of reference drug was added to formulate tablets then the estimation of nominal value was done. Repeatation was done for three times.

Precision:

Repeatability of method was established by analyzing various replicate standards of mefenamic acid. Results for the repeatability were found within limit. All the solutions were analyzed thrice, in order to record any intra-day and inter-day variation in the result.

Robustness:

Robustness of the method was determined by carrying out small deliberate variations in the optimized method parameters. The effect of variations of mobile phase pH, flow rate, chromatographic system, operator and column temperature on the retention time and tailing factor were studied.

Specificity:

A blend of commonly used excipients such as methyl paraben, propyl paraben, gum acacia, talc, lactose and starch was treated with mefenamic acid and then assayed as per developed procedure.

RESULTS AND DISCUSSION:

Different mobile phases were tried by changing proportion of organic phase (Acetonitrile) at different pH ranges. The mobile phase was optimized on the basis of asymmetry factor, peak area obtained, retention time and number of theoretical plates. Amongst the various compositions tried; satisfactory separation, well resolved, short retention time, and good symmetrical peaks were obtained with the mobile phase comprising Acetonitrile : water (80:20) at pH 3.0 adjusted with glacial acetic acid. The UV-Vis scan of mefenamic acid in mobile phase revealed absorption maximum at 288 nm. Hence this wavelength was selected for the further analysis. The scan is shown in Figure 1.

Figure 1: The UV spectra of mefenamic acid in the mobile phase

The peaks were highly resolved and shown in Figure 2. The retention time, the asymmetry factor and number of theoretical plates for Mefenamic acid and Diclofenac sodium are depicted in TABLE 1.

Figure 2: Chromatogram of mixture of mefenamic acid and diclofenac sodium

TABLE 1: System suitability parameters for mefenamic acid and diclofenac sodium

Parameters	Mefenamic acid	Diclofenac sodium
Retention time (min.) ± SD	3.133 ± 0.042919	2.750 ± 0.039177
Width	0.110	0.101
Area (µV.sec) ± SD	1336819.1372 ± 4780.760	619713.7615 ± 5828.2582
% Area	68.326	31.674
Plates	4461.81	4080.67
Capacity	375	329
Resolution	2.135	0.000
Asymmetry	1.331	1.467

TABLE 2: Linearity of mefenamic acid

Standard Conc.	10 μg/ml	30 μg/ml	50 μg/ml	70 μg/ml	90 μg/ml
Replicates	Peak area (µV.sec)
1	248169.3	823515.5	1431915	1979911	2592589.96
2	247352.9	828723.7	1429651	1989739	2599625.74
3	246849.7	826548.9	1436934	1981826	2581974.86
4	248658.3	823217.3	1439675	1986296	2587986.28
5	249172.6	821842.2	1448721	1996982	2574392.63
Mean	248040.6	824769.5	1437379	1986951	2587313.89
SD	944.8235	2799.713	7478.257	6794.453	9686.77873
% RSD	0.380915	0.339454	0.52027	0.341954	0.37439519

TABLE 3: Recovery studies and Statistical analysis of results

Formulation	Label claim(mg)	% Estimated	COV (%)	S E*	95% Confidence interval	%Recovery*
MEFTAL	250	100.05204	1.109855	0.641108	2.758689	98.909637
PONSTAN	250	100.350769	1.211601	0.701972	3.020584	99.228737
MEFLUP	250	100.191948	1.101504	0.637174	2.741762	99.898865

* Mean of three determinations

SD- Standard deviation, COV- Coefficient of variation, SE- Standard error

TABLE 4: Intra-day variability of mefenamic acid

Concentration (μg/ml)	Peak area (µV.sec)			Mean area (µV.sec)	SD	% RSD
Concentration (μg/ml)	Trial 1	Trial 2	Trial 3	Mean area (µV.sec)	SD	% RSD
10	248169.3	246221.5	247193.4	247194.7	973.8977	0.39398
20	572755	570973.4	575759.6	573162.7	2419.021	0.422048
30	823515.5	826941.7	828472.7	826310	2538.246	0.307178

TABLE 5: Inter-day variability of mefenamic Acid

Concentration (μg/ml)	Peak area (µV.sec)			Mean area (µV.sec)	SD	% RSD
Concentration (μg/ml)	Day 1	Day 2	Day3	Mean area (µV.sec)	SD	% RSD
30	823515.5	820397.3	825348.9	823087.2	2503.439	0.304152
40	1088905	1073535	1083871	1082104	7836.212	0.724165
50	1431915	1437925	1455232	1441690	12106.23	0.839724

The calibration plot is shown in Figure 3. The peak areas for the corresponding concentrations of the calibration curve are depicted in TABLE 2. The standard deviations for all concentration levels were very low and the % RSD also did not exceed 0.4 %. The coefficient of correlation was highly significant which was 0.99965 with 0.296482 % COV. The linearity range was observed between 0-90 μg/ml. The plot clearly showed a straight line (y = 28998.214844x - 36339.367188).

Figure 3: Calibration curve for mefenamic acid

The accuracy of the method was judged by recovery studies. The results are shown in TABLE 3. The precision of the method was evaluated by intraday and interday analysis which also showed good results with very low variations as revealed by very low % RSD values which are shown in TABLE 4 and 5.

The LOD and LOQ value for Mefenamic acid was found to be 0.3137 μg/ml and 0.9508 μg/ml, which suggests that a nanogram quantity of mefenamic acid can be estimated accurately.

The proposed HPLC method was compared with official titration method. The results obtained showed that the calculated t- and F- values did not exceed the theoretical values (95% confidence limits for five degree of freedom), shown in TABLE 6, from which we can conclude that the proposed methods do not differ significantly from reference method.

TABLE 6: Statistical analysis of mefenamic acid using the HPLC method compared with official method

Analytical method	Mefenamic acid
Analytical method	Mean ± SD	T	F
HPLC method*	100.05 ± 0.9588	1.174 (2.353)	2.745 (3.62)
Official method*	99.972 ± 0.09931

*Each value is average ± SD (n=5)

CONCLUSION:

The present HPLC method was found to be simple, accurate, reproducible and rapid, and can be successfully employed in routine analysis in quality control laboratories for different type of pharmaceutical formulations.

ACKNOWLEDGEMENT:

Authors are grateful to Ajanta Pharma Ltd., Mumbai and Bombay Drug House, Mumbai for providing the gift samples. We are also thankful to the principal and management of Appasaheb Birnale College of Pharmacy, Sangli for providing the necessary facilities to carry out this work.

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Received on 18.03.2010 Modified on 01.04.2010

Asian J. Research Chem. 3(3): July- Sept. 2010; Page 691-694