Validation of a Spectrophotometric Method for the Determination of Mefenamic Acid in Pharmaceutical Formulations
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1 doi: /dac Validation of a Spectrophotometric Method for the Determination of Mefenamic Acid in Pharmaceutical Formulations Walaa A.A. Alballaa 1, Ayman A.A. Ali 1, Omer A.A. Hamdi 1, Abubakr M. Idris 2 1Department of Chemistry, Faculty of Science and Technology, Alneelain University, 11121, Khartoum, Sudan 2Department of Chemistry, College of Science, King Khalid University, Saudi Arabia jacknon@hotmail.com Abstract A simple, sensitive and specific UV-Spectrophotometric method was developed for the determination of Mefenamic acid in tablets dosage form. The method is based on the oxidation of mefenamic acid with iron (III), and subsequent complexation of iron (II) with o-phenanthroline to produce a red colored complex (ferroin), which was monitored at 510 nm. The variables affecting oxidation of drug were studied and optimized. Under the experimental conditions used, the calibration graphs were linear over the range µg/ml, the molar absorptivity and sandal's sensitivity were L.mol-1.cm Keywords Spectrophotometric; Validation Method; Mefenamic Acid Introduction Mefenamic acid (MFA), as anthranilic acid derivative, is chemically named [2-(2,3-dimethylphenyl)amino]benzoic acid (Fig. 1). MFA has showed anti-inflammatory properties with analgesic and antipyretic effects (Espinosa et al, 2005). It is used specially in the treatment of rheumatoid arthritis, osteoarthritis and other muscular-skeletal diseases (Espinosa et al, 2005). COOH CH 3 N CH 3 H FIG. 1 CHEMICAL STRUCTURE OF MEFENAMIC ADID MFA has also been found to be effective to produce closure of patent ductus arteriosus in premature neonates (Espinosa et al, 2005). Due to its wide range of therapeutic activities have been reported. Among those methods, titrimetry (Cakyrer et al, 1999), spectrophotometry (Maron et al, 1990; Aboul Khieret al, 1987; Dinc et al, 2002), luminescence (Arnaud et al, 2003; Aly et al, 2000), electrophoresis (Perez-Ruiz et al, 1998; Polasek et al, 2000) and chromatography (Hilton et al, 2003; Rouini et al, 2004; Sun et al, 2003; Mikami et al, 2000; Hirai et al, 1997) have been reported (Albero et al, 1995). In the present work, a new, simple and sensitive UV-spectrophotometric method is described for the assay of mefenamic acid in tablet dosage form. The method is based on oxidation of MFA with iron (III), and subsequent complexation of iron (II) with o-phenanthroline to produce a red colored complex (ferroin). The variables affecting the oxidation of the drug were studied and optimized. 12
2 Development in Analytical Chemistry (DAC) Volume 3, Experimental Section Materials MFA standard and mefenamic acid tablets 500mg were obtained from Wafra pharma LDT, Khartoum, Sudan. All chemicals used of analytical or pharmaceutical grade were dissolved using distilled water. Instrumentation v UV/Vis double beam Spectrophotometer/800 (Shimadzu, Japan) with matched quartz cell (1 cm) was used in this study. Spectrum scan was performed for 20 µg/ml MFA in the UV range of nm. Methods 1) Preparation of Standard and Sample Solution 0.1 g of MFA was gradually weighed, then, it was dissolved in 100 ml of 0.01 M sodium hydroxide. In 1 L volumetric flask, the volume was completed by distilled water up to mark. 2) Sample Treatment 10 tablets were gradually weighed and then they were powdered. A quantity of the powdered tablets containing 0.1 g of MFA was dissolved in 100 ml of 0.01 M sodium hydroxide. 3) Preparations of Buffer Solutions Different chemicals (potassium chloride, potassium hydrogen phthalate, potassium dihydrogen phosphate, boric acid, sodium bicarbonate and hydrogen hydroxide) were used to prepare buffer solutions with different ph range from 1 to 13. Data Analysis 1) Validation of the Development Methods The development methods for estimation of MFA were validated as per ICH guidelines [ICH, 1996]. 2) Linearity and Regression Indicates the ability to produce results that are directly proportional to the concentration of the analyte in samples. A series of samples were prepared in which the analyte concentrations span the claimed range of the procedure. If there is a linear relationship, the results test was evaluated by appropriate statistical methods. Absorbance was observed for five samples with different concentrations (0.1, 0.09, 0.08, 0.07 and 0.06 μg/ml). Regression of linearity is a statistical analysis assessing the association between tow variables, it is used to find the relationship between tow variables, and according to equation of regression: Y= a + b x Where Y practical response, a intercept, b slope, and x concentration of the sample. 3) Accuracy (Recovery) Accuracy of the method was studied by recovering experiments using the standard addition method at nine different levels. Known amounts of standard solutions containing mefenamic acid (9.2, 9.4, 9.6, 1.22, 1.24, 1.28 and 1.52 μg) were added to prequantified sample solution. These samples were analyzed and recovery was calculated. 4) Specificity (Selectivity) All weights were transferred to 100 ml volumetric flask, 30 ml of dissolution media was added, then it needed to dissolve at room temperature. After need that dissolution media volume was completed, 10 ml was transferred to 100 ml volumetric flask for standard test and placebo solutions, then the volume was completed with dissolution media. 13
3 The absorption was observed only in the pure mefenamic acid and does not absorption have been observed in the additive (placebo) and in paracetamol. 5) Range The range for assay method was demonstrated by analyzing placebo solutions in the range between 50 % to 150 %of nominal concentration. Three weights were prepared at different concentrations from 50 % to 150 %. Each of one had been read three times. Precision 1) Intermediate Precision Samples of the mefenamic acid were recorded during the day at different times (0 minutes, 1 minute, 5 minutes and 10 minutes) respectively. 2) Repeatability Repeatability is given by inter-day and intra-day precision. Intra-day precision was determined by analyzing the three different concentration of mefenamic acid for three times in the same day. Inter-day precision was determined by analyzing the three different concentration of mefenamic acid for three days in a week. 3) Robustness The analytical procedure of acceptable accuracy and precision under a variety of conditions was provided. The results from separate samples were influenced by changes in the operational or environmental conditions. Robustness of mefenamic acid showed different concentrations at different temperatures (65, 67, 70, 73 and 75 C) and at different wavelengths (512, 511, 510, 509 and 508 nm). 4) Ruggedness Different concentrations of mefenamic acid samples and standard mefenamic acid were observed by dissolved them in four different solutions with different concentrations (NaOH 0.1 M, NaOH 0.05 M, KOH 0.2 M and HCl 0.1 M). Results and Discussion Primary Study The spectrum of MFA was scanned in the maximum absorbance at 510 nm. The studied reactions in this method involved oxidation reaction and complexation reaction. MFA was first oxidized by iron (III). Subsequently, the product iron (II) reacts with o-phenanthroline to produce a red colored complex (ferroin) as obtained in Figure (2), which was monitored at 510 nm. FIG. 2 FERRION COMPLEX FORMATION 14
4 Concentration Development in Analytical Chemistry (DAC) Volume 3, Method Validation Linearity and Regression A set of standard solutions of MFA was subjected to the proposed method. Acceptable linearity range ( µg/ml) with correlation coefficient was obtained in Table (1). Each concentration was applied in triplicate. The Correlation coefficient ( ) showed good similarity to that reported by Teena et al, (2014), it was TABLE1. THE LINEARITY SITED OF MFA SAMPLES Conc. (μg/ml) Abs Comments The correlation coefficient was Slope Intercept y = x R² = Absorbance FIG. 3 LINEARITY OF MEFENAMIC ACID 2) Accuracy Accuracy of MFA samples was illustrated in Table (2). The results referred to the average of three assays for each concentration. The results are in good agreement with acceptable values for the validation of an analytical procedure. The measured concentration of MFA was increased by increasing the MFA amount added, the average value of recovery was (99.29 ± 0.1), similar to that reported by Teena et al, (2014), it was 98.8 ±
5 TABLE2. ACCURACY (RECOVERY) OF MFA SAMPLES MFA amount added (μg) Conc Recovery ± ± ± ± ± ± ± ± ± 0.8 The selectivity of the method is presented in Table (3); the concentration of MFA samples was µg/ml, but the concentration of paracetamol samples and placebo was zero, because they don t absorb at 510 nm. The recovery values of MFA in the presence of excipients and acetaminophen, is presented in Table (4); the excipients (Talc, Mg-stearate, Maize-starch, corn-starch, microcrystalline cellulose, hydroxyl propyl cellulose (HPC), Lactose monohydrate and AVCL 101) seem to not affect the average recovery. 3) Range The range of MFA for assay method was demonstrated by analyzing placebo solutions, the range of mefenamic acid between 50% and 150%. In Table (5), the concentrations of mefenamic acid were increased when range increased. MFA has recorded the highest concentration µg/ml at range concentration 150%. Precision 1) Intermediate Precision MFA sample with three replications was read at different times (1 minute, 5 minutes and 10 minutes) respectively. The results from intermediate precision were presented in Table (6). The concentrations of MFA of the three replicates was µg/ml. The precision of MFA was 99.51, it more than that reported by Teena et al, (2014), it was ± ) Robustness In both Tables (7) and Table (8), the robustness of the proposed method was examined at different temperatures and different wavelengths. Table (7) showed the concentration of MFA at five levels of temperatures (65, 67, 70, 73 and 75 C). The percentage of MFA in different temperatures was increased b y increase the temperature. The robustness percentage were showed similarity to that reported by Teena et al, (2014), there were ± 1.27, ± 1.28, ± 0.87 and ± Table (8) illustrated concentration of MFA at five levels of wavelengths (508, 509, 510, 511 and 512 nm), the concentration of MFA was increased by increase the wavelengths. 3) Ruggedness Different concentrations of mefenamic acid samples and standard mefenamic acid were observed by dissolved them in four different solutions with different concentrations (NaOH 0.1 M, NaOH 0.05 M, KOH 0.2 M and HCl 0.1 M), the results were tabulated in Table (9). The concentration of MFA was more in NaOH 0.1 M solution, but it neared to zero in HCl medium for the acidity nature of MFA. The ruggedness percentage of MFA samples was more than that reported by Teena et al, (2014), there were ± 1.96, ± 1.10 and ± ) Repeatability The results of repeatability of MFA samples were presented in Table (10). The results were illustrated excellent % RSD less than 2% 16
6 Development in Analytical Chemistry (DAC) Volume 3, TABLE 3. SPECIFICITY (SELECTIVITY) OF MFA, PARACETAMOL SAMPLES AND PLACEBO No Sample ID at wavelength 510 Conc of sample Conc of standard Percentage Type nm 1 Mefenamic-Avg Average (n = 3) Paracetamol-Avg Average (n = 3) plac-avg Average (n = 3) TABLE 4. DETERMINATION OF MFA SAMPLES IN PRESENCE OF EXCIPIENTS Excipients Amount taken Average recovery Talc Mg-stearate Maize-starch Corn-starch Microcrystalline cellulose Hydroxy propyl cellulose (HPC) Lactose monohydrate AVCL TABLE 5. RANGE OF MFA SAMPLES Theoretical concentration 50 % 75 % 100 % 125 % 150 % Measured concentration A B C Average STDV RSD TABLE 6. INTERMEDIATE PRECISION OF MFA SAMPLES No Sample ID at wavelength at 510 Conc of sample Conc of standard Percentage Type nm 1 Mefenamic-Avg Average (n = 3) (1min)-Avg Average (n = 3) (5min)-Avg Average (n = 3) (10min)-Avg Average (n = 3) No Sample ID Type TABLE 7. ROBUSTNESS OF MFA SAMPLES IN DIFFERENT TEMPERATURES Conc of sample Conc of standard Percentage 1 Mefenamic (65)-Avg Average (n = 3) Mefenamic (67)-Avg Average (n = 3) Mefenamic (70)-Avg Average(n = 3) Mefenamic (73)-Avg Average(n = 3) Mefenamic (75)-Avg Average(n = 3) TABLE 8. ROBUSTNESS OF MFA SAMPLES IN DIFFERENT WAVELENGTHS No Sample ID Type Wavelength (nm) Conc of sample Conc of standard 1 Mefenamic (512)-Avg Average (n = 3) Mefenamic (511)-Avg Average (n = 3) Mefenamic (510)-Avg Average (n = 3) Mefenamic (509)-Avg Average (n = 3) Mefenamic (508)-Avg Average (n = 3)
7 TABLE 9. RUGGEDNESS OF MFA SAMPLES No Sample ID at wavelength 510 Conc of sample Conc of standard Percentage Type nm 1 NaOH (0.1 M)-Avg Average (n = 3) NaOH (0.05 M)-Avg Average (n = 3) KOH (0.2 M)-Avg Average (n = 3) HCl (0.1 M)-Avg Average (n = 3) TABLE 10. REPEATABILITY OF MFA SAMPLES Drug Mefenamic acid Amount taken Inter day Amount found RSD Intra day Amount found RSD Conclusions The proposed method represents a promising approach in the area of pharmaceutical monitoring with low cost, high speed, simplicity and sensitivity and therefore can be recommended for the routine analysis of the drug in quality control laboratories. The proposed method showed good accuracy and repeatability for determination of drug in pharmaceutical dosage form. The sensitivity of the proposed method is comparable or better than other titration methods with wider linear range. REFERENCES [1] AboulKhier A.; El-Sadek M.; Baraka M. Analyst (1987), 112, [2] Albero M. I.; Sanchez-Pedreo C.; Garcia M. S. J. Pharm. Biomed. Anal. (1995), 13, [3] Aly F.A.; Al-TamimiS. A.; Alwarthan A. A., Anal. Chim. Acta (2000), 416, 87. [4] Arnaud N.; Georges. J. Anal. Chim. Acta (2003), 467, 149. [5] Cakyrer O.; Klç E.; Atakol, O.; Kenar A. J. Pharm. Biomed. Anal. (1999), 20, 19. [6] Dinç E.; Yücesoy C.; Onur F. J. Pharm. Biomed. Anal. (2002), 28, [7] Espinosa-Mansilla A.; Munoz de la Pena A.; Canda-Canda F.; Gonzez-Gmez D. Anal. Biochem. (2005), 37, 275. [8] Fouad F.Q, Abdullah M.P., Rozali M.O., Wan M.A.W. Int. J. Chem. Sci.: 12(1), 2014, [9] Hilton M. J.; Thomas K. V. J. Chromatogr. A (2003), 1015, 129. [10] Hirai T.; Matsumoto S.; Kishi I. J. Chromatogr. B (1997), 692, 375. [11] Maron, M.; Wright, G. J. Pharm. Biomed. Anal. (1990), 8, 101. [12] Mikami E.; Goto T.; Ohno T.; Matsumoto H.; Inagaki K.; Ishihara H.; Nishida M. J. Chromatogr. B (2000), 744, 81. [13] Perez-Ruiz T.; Martnez-Lozano C.; Sanz A.; Bravo E. J. Chromatogr. B (1998), 708, 249. [14] Polasek M.; Pospisilova M.; Urbanek M. J. Pharm. Biomed. Anal. (2000), 23, 135. [15] Rouini M. R.; Asadipour A.; Hoseinzadeh Y.; Aghdasi F. J. Chromatogr. B (2004), 800, 189. [16] Siladity B., Subhajit G., Fahad A., Saayak S. and Sritoma B. J Anal Bioenal Techniques (2012), 3, 6. [17] Sun, Y.; Takabe, K.; Kido H.; Nakashima M. N.; Nakashima K. J. Pharm. Biomed. Anal. (2). [18] Teena O., Suryakant B., Sonalinak. I J PSR (2014), 5, 6. 18
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