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Available online at www.pharmscidirect.com Int J Pharm Biomed Res 2011, 2(1), 26-29
International Journal of PHARMACEUTICAL AND BIOMEDICAL RESEARCH ISSN No: 0976-0350
Research article
Spectrophotometric determination of tranexamic acid and mefenamic acid in tablet dosage form using derivatization technique N. Subramanian*, T. Devipriyadharshini, K. Venkateshwaran, P. Chandrasekar Laboratory for Lipid Based Systems, Department of Pharmaceutical Technology, Anna University of Technology, Tiruchirappalli- 620 024, Tamilnadu, India.
Received: 25 Jan 2011 / Revised: 29 Jan 2011 / Accepted: 03 Feb 2011 / Online publication: 20 Feb 2011
ABSTRACT A new effective UV spectrophotometric method has been developed for the simultaneous estimation of tranexamic acid and mefenamic acid in combined tablet dosage form based on derivatization technique for making tranexamic acid UV detectable employing reaction of ninhydrin with primary amino group of tranexamic acid in the presence of basic medium via oxidation, deamination followed by condensation to form ruhemann purple product, quantitative determination is performed by measuring the absorbance at two different wavelengths 571 nm and 335 nm. Beer’s law is obeyed in the concentration ranging from 8- 40 μg/mL for tranexamic acid and 4- 20 μg/mL for mefenamic acid. The proposed method is validated for linearity, recovery, precision, robustness and specificity by statistical evaluation as per ICH guidelines. Tranexamic acid and mefenamic acid calibration curves are linear with correlation coefficient of 0.9983 and 0.9991. Recovery is between 98.0% - 101.0% for both tranexamic acid and mefenamic acid. The developed method can be used for routine quality control analysis of titled drugs combination in tablet formulation. Key words: Mefenamic acid, Tranexamic acid, Ninhydrin, Method validation, Simultaneous equation method
1. INTRODUCTION Tranexamic acid [Fig.1A], chemically known as trans- 4aminomethyl-cyclohexacarboxylic acid [1], is the most potent antifibrinolytic agent that competitively inhibits activation of plasminogen to plasmin (fibrinolysin), an enzyme that degrades fibrin clot, fibrinogen, and other plasma proteins [2]. It is used in treatment of pre and postoperative conditions, bleeding disorders (menstrual blood loss and bleeding during pregnancy) and during and following tooth extraction of hemophilic patient [3, 4]. It also acts as adjuvant drug for site specific pharmaco laser therapy [3]. Tranexamic acid lacks π-electrons to behave as a chromophore, to enable direct analysis and detectable by UVvisible absorption spectrophotometry, hence it is essential to make derivatives of tranexamic acid [5] and its structure shares similarity to aminoacids with its amino and carboxylic acid groups. *Corresponding Author. Tel: +91 431 2407978, Fax: +91 431 2407333 Email:
[email protected]
©2011 PharmSciDirect Publications. All rights reserved.
Mefenamic acid [Fig.1B], chemically N-[(2, 3-dimethyl phenyl) amino] benzoic acid [6], is the derivative of Nphenylanthranilic acid and official in USP, BP and IP [7]. It has potent anti-inflammatory drug, analgesic and antipyretic properties [6]. Mefenamic acid shows preferential inhibition of cyclooxygenase- 2 there by inhibits the synthesis of prostaglandins [8]. It is used in the treatment of primary dysmenorrheal, osteoarthritis, nonarticular rheumatism, healing of wounds, sport injuries [6], periodontitis, antiphlogistic, rheumatoid arthritis [9] and other painful musculoskeletal illnesses [10]. Trends in pharmaceutical analysis and compendial assay methods reflects increasing replacement of time- honoured classical methods of analysis with modern sophisticated instrumental methods, like HPLC, LC- MS/MS. However, due to high acquisition and maintenance cost of this equipment makes adoption of an alternative and equivalent UV method. The British Pharmacopoeia also gives allowance for the use of an alternative method for any test or assay. New tablet formulation in combination of tranexamic acid 500 mg and mefenamic acid 250 mg is commercially available in Indian market for treatment of menorrhogia [4]. Few chromatographic and spectrophotometric methods are
N. Suubramanian et al.., Int J Pharm Bio omed Res 2011, 2(1), 2 26-29
reeported for the determinnation of traanexamic aciid and m mefenamic aciid individuallly or in com mbination withh other drrug. The repoorted methodss for the estim mation of mefeenamic accid was carrieed out by usinng different annalytical instruuments like liquid chrromatographyy- tandem MS S [11], HPLC C [12], coonductometerr [10], protonn NMR [10],, spectrophotoometer [113], gas liquiid chromatogrraphy [9] andd spectrofluorrimeter [110] where ass tranexamic acid was deetermined byy using pootentiometer [14], spectrrofluorimeter [14], electrrospray liquid ioonization m mass specctrophotometeer [1], chhromatographhy tandem maass spectrophootometer [11],, TLC deensitometer [14] [ and specctrophotometeer [4]. Howevver, so faar no reporrt has been found for the simultaaneous deetermination of tranexamiic acid and mefenamic acid a in phharmaceuticall preparationss using UV sppectrophotomeeter up too best of our knowledge. k Hence thee present stuudy aimed to t develop an a UV sppectrophotom metric method for the simuultaneous estim mation off tranexamic acid and mefenamic acid in combinedd tablet doosage form baased on derivaatization technnique. 2.. MATERIAL LS AND METHODS 2..1 Chemicals was obtainned Zota Tranexamiic acid from Pharmaceuticalls, Chennai; mefenamic acid was obbtained frrom Fourrts India Pvt. Ltd, Chennaai. Ninhydrinn was puurchased from m Merck, Mumbai. M Tableets were purrchased frrom Indian market, m containning 500 mg of tranexamiic acid annd 250 mg off mefenamic acid a per tableet. Methanol (HPLC ( grrade) was purrchased from SD S Fine Chem micals Ltd, Muumbai. 2..2 Apparatus Analysis was w performeed on a Shim madzu doublee beam U UV–visible spectrophotom meter equipped with 100 mm m matched quartzz cell model UV U 1800 (Japaan). 2..3 Standard preparation annd derivatizatiion Standard stock s solutionns of tranexam mic acid (25 µg/mL) µ annd mefenam mic acid (12.5 µg/mL) were prepared by trransferring 400 mg of traanexamic aciid and 20 mg m of m mefenamic aciid in to a 1000 mL volumettric flask conttaining 200 mL diluent (methanol: pH p 7.2 phosphhate buffer, 70 7 : 30, v//v). It was thhen sonicatedd for 15 minn. The solutioon was diiluted up to voolume with diiluent. Series of working sttandard soolutions were prepared. To the 1 mL worrking standardd 2 mL off ninhydrin solution s (0.2 % in methannol) was addeed and m mixed well [4]. The mixturee was warmedd for 20 min at a 80°C using constant temperature bath b [3] and then t cooled too room teemperature finnally volume was made upp to the markk using diiluent.
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(A) (B) g.1. Chemical struucture of Tranexaamic acid (A) and d Mefenamic acidd (B) Fig
2.4 4 Sample prepparation and dderivatization From the trriturates of 20 tablets an accurately a weeighed po ortion of pow wder (45.0 m mg) was transsferred to 1000 mL vo olumetric flaskk and mixed w with 70 mL of diluent, sonicated forr 15 min; the solution was made up to volume v with diluent d an nd filtered throough 0.45 µm membrane fillter [15]. Aliqquot (1 mL L) of the filtrrate was derivvatized using 2 mL of ninhhydrin (0..2 % in metthanol) warm med at 80°C for 20 min using constant temperrature bath, ccooled, cooleed made up to t the maark using diluuent [16,17]. T The absorban nce of standarrd and sam mple was meeasured at dual mode (571 1 nm and 3355 nm) ag gainst ninhydriin blank. 2.5 5 Simultaneouus equations (M (Multi-compon nent method) The wavellengths seleccted for solv ving simultaaneous eq quation are 5771 nm and 3335 nm (Tablle 1). Absorpptivity coefficients off both drugs were determ mined at selected waavelengths usiing simultaneoous equations 1 and 2. [7]. Cx x = (A2 ay1 - A1 ay2)/ (ax22 ay1 - ax1 ay2) ------------- (1) Cy y = (A1 ax2 - A2ax1)/ (ax2 ay1 - ax1 ay2 2) --------------- (2) wh here, A1 and A2 are the abbsorbance of sample s solutioons at 56 68nm and 334nm resppectively. Cx C and Cyy are concentrations of tranexam mic acid and d mefenamic acid sam mple solutionn. By substitutting the valuess of A1 and A2 A and solving the twoo equations siimultaneously y, the values of Cx an nd Cy can be b obtained. Here, ax1 and ax2 are the absorptivity coeefficient of traanexamic acid d at 571nm annd 335 nm m respectivelyy, ay1 and ay22 are the abso orptivity coeffficient off mefenamic accid at 571nm and 335 nm reespectively. 2.6 6 Method deveelopment The solubillity of tranexaamic acid and mefenamic acid a in diffferent buffeers like pH 5.5, 6.0, 7.2 and vaarious combination of methanol andd buffer pH 7.2 7 was determ mined. Diiluents containning phosphatte buffer pH 7.2 7 and methaanol in thee ratio of (330:70 v/v) ussed to record d the spectruum of traanexamic acidd and mefenaamic acid in the wavelenggth of 57 71nm for traneexamic acid annd 335nm for mefenamic accid.
N. Subramanian et al., Int J Pharm Biomed Res 2011, 2(1), 26-29 Table 1 Absorptivity for tranexamic acid and mefenamic acid Concentration (μg/mL) Absorptivity Tranexamic acid/ at 571 nm at 335 nm mefenamic acid Tranexamic Mefenamic Tranexamic acid acid acid 8/4 2.47 0.005 0.458 16/8 2.40 0.008 0.466 24/12 2.28 0.010 0.410 2.30 0.021 0.399 32/16 40/20 2.23 0.025 0.341 Mean 2.33 0.014 0.414 SD 0.000 0.000 0.0002
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2.7.3 System and method precision
Mefenamic acid 2.50 2.31 2.23 2.18 2.17 2.28 0.0001
Precision of an analytical procedure expresses the closeness of agreement between a series of measurements obtained from multiple sampling of same homogenous sample under prescribed conditions. Method precision was determined by analyzing the standard working solution at five times [21]. System precision was determined by five replicate measurements of working sample mixture at analytical concentrations. 2.7.4 Ruggedness (Inter- intra day and analyst variations)
Table 2 Optical parameters for tranexamic acid and mefenamic acid Parameters studied Tranexamic acid S. No 1 Beers law (μg/mL) 8- 40 2 Slope 0.023 Intercept 0.017 3 4 R2 0.999 λ max(nm) 571 5
Mefenamic acid 4- 20 0.031 0.012 0.998 335
The intra-day (repeatability), inter-day (intermediate precision) variability and analyst to analyst variations were determined using standard solutions [21]. These experiments were repeated at different times on the same day to evaluate intra-day variability, on second day to evaluate day- day variability and by different analyst to evaluate analyst variation. 2.7.5 Robustness
y = 0.031x + 0.012 R² = 0.998
1.2 MEFENAMIC ACID 1
TRANEXAMIC ACID y = 0.023x + 0.017 R² = 0.999
Absorbance
0.8
Robustness of the method was determined by small deliberate variations of the analytical method parameters [19]. The condition studied is altered wavelength of 571±3 nm for tranexamic acid and 335±3 nm for mefenamic acid.
0.6
3. RESULTS AND DISCUSSION
0.4
The present UV spectrophotometric method has been developed for the simultaneous estimation of tranexamic acid and mefenamic acid in pharmaceutical formulation using common conditions and to be used in routine analysis. Tranexamic acid is soluble in water whereas mefenamic acid is insoluble in water but soluble in alcohol. Mefenamic acid is also shown pH dependent solubility (data not shown). The common solvent for these drugs was selected by dissolving the drugs in phosphate buffers of pH 5.5, 6.0 and 7.2 and in various combination of methanol and phosphate buffer pH 7.2. The combination of methanol and phosphate buffer pH 7.2 in the ratio of 70:30 has shown highest solubility and clear spectrum for the both drugs. Ninhydrin (0.2 % in methanol) was used as derivatization agent for the tranexamic acid. Cross wavelength study was performed to determine the absorbance of the both drugs at 571nm and 335nm. The absorptivity coefficient for the both drugs is determined by using Beer’s law and the content of tranexamic acid and mefenamic acid was calculated using simultaneous equations 3 and 4.
0.2 0 0
4
8
12
16
20
24
28
32
36
40
44
48
Concentration (mcg/mL)
Fig.2. Linearity of Tranexamic acid and Mefenamic acid
2.7 Method validation 2.7.1 Linearity Linearity was determined by appropriate dilutions of standard stock solutions [18] of tranexamic acid and mefenamic acid ranging from 8 µg/mL to 40 µg/mL for tranexamic acid and 4 µg/mL to 20 µg/mL for mefenamic acid. The linearity of the working standard solution was obtained by plotting the absorbance versus concentrations of respective drugs shown in Fig.2. 2.7.2 Recovery Recovery of the method was calculated by standard addition at three levels like low, middle and high (80%, 100% and 120%) concentrations [19, 20] in triplicates.
Cx= (0.3744×0.014) - (0.5503×2.228) / (0.414×0.014) (2.233×2.28) ------------ (3) Cy= (0.5503×0.414) - (0.3744×2.33) / (0.414×0.014) (2.233×2.28) ------------ (4)
N. Subramanian et al., Int J Pharm Biomed Res 2011, 2(1), 26-29 Table 3 Method validation parameters for tranexamic acid and mefenamic acid Parameters Tranexamic acid RSD (%) of absorbance Recovery 0.9 80 0.2 100 0.1 120 Precision 1.8 System precision Method precision 0.2 Variations Analyst-1 variation 0.9 Analyst-2 variation 0.4 Inter day variation 0.7 Intra day variation 1.0 Robustness (altered wavelength) 567nm/332nm 0.1 570nm/335nm 1.2 573nm/338nm 1.1 RSD- Relative standard deviation
The absorptivity coefficients of tranexamic acid at 571nm and 335nm was found to be 2.33 and 0.414 and for mefenamic acid was found to be 0.014 and 2.28. The optical parameters are shown in Table 2 which shows an excellent correlation between absorbance and concentration of each drug within the concentration tested. The correlation coefficient (R2) values for both the drugs were found to be >0.996. The mean percentage recoveries obtained for tranexamic acid and mefenamic acid as shown in Table 3 are closer to 100% indicates non- interference from the excipients used in the formulation. The % RSD of five replicate analysis for method precision, system precision, intra- inter day and analyst variations are found to be less than 1.0 %. The % RSD for the deliberate change in the dual wavelength for both drugs is found to be less than 2.0 %. The observed results indicate the method to be specific, accurate, precise and reproducible. 4. CONCLUSIONS Proposed method has the advantage of simplicity, convenience, precise and accurate. Ninhydrin provides a simple reagent for spectrophotometric determination [22] of tranexamic acid and mefenamic acid due to its high sensitivity and low absorbance of ninhydrin blank. Moreover the developed method does not involve any stringent reaction conditions and offers advantage of color stability. Therefore this method can be successfully applied for the determination of tranexamic acid and mefenamic acid in tablet dosage form.
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Recovered (%)
Mefenamic acid RSD (%) of absorbance
Recovered (%)
99.2 98.7 98.2
1.0 0.7 0.7
99.4 99.5 100.1
2.0 0.7 0.7 0.1 1.1 1.3 0.8 0.7 1.1
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