Development of spectrophotometric method for determination of mesalazine in pharmaceutical preparation - Nguyen Trung Dung

Vietnam Journal of Chemistry, International Edition, 54(4): 509-514, 2016 DOI: 10.15625/0866-7144.2016-00356 509 Development of spectrophotometric method for determination of mesalazine in pharmaceutical preparation Nguyen Trung Dung * , Nguyen Thi Lan Anh, Dong Thi Oanh, Nguyen Hoang Dung Faculty of Physics and chemical Engineering, Le Quy Don Technical University Received 18 May 2016; Accepted for publication 12 August 2016 Abstract A simple, rapid, sensitive and accurate spectrophotometric method for the determination of mesalazine in pharmaceutical preparation has been developed. The method is based on the reaction of mesalizine, with excessive nitrite, in an acidic medium, to produce the corresponding diazonium salt. After the removal of residual nitrite with sulphamic acid, the diazonium salt was coupled with N-(1-naphthyl)ethylenediamine (NEDA) in acidic medium to give a violet colored azo dye which has maximum absorption at 552 nm, the colored chromogen was stable for twenty four hours. Beer's law is obeyed in the concentration range of 0.5-40.0 µg/ml (R 2 = 0.999) with limit of detection (LOD) being 0.113 µg/ml. The molar absorptivities were 1.5.10 4 l.mol -1 .cm -1 and Sandell's sensitivity index of 0.0263 μg.cm-2. Recoveries of mesalazine from excipients were 95.4 -102.3 %. No interference was observed from common excipients present in pharmaceutical formulations. The proposed method has been applied successfully to determine mesalazine in pharmaceutical preparation as tablets (Pentasa and Sunmesacol). Keywords. Mesalazine, spectrophotometry, NEDA, Bratton-Marshall reagent, Pharmaceutical Preparation. 1. INTRODUCTION Mesalazine (mesalamine, 5-aminosalicylic acid, 5-ASA) is used to treat inflammatory bowel diseases, especially non-specific ulcerative colitis and Crohn’s disease. Drugs based on both mesalazine parent substance and its prodrugs (sulfasalazine, olsalazine, balsalazide) are often used for this. Mesalazine is metabolized in vivo by acetylating enzymes that produce N- acetylmesalazine (N-acetyl-5-aminosalicylic acid) (Fig. 1) [1, 2]. A number of analytical methods have been reported for the determination of 5-ASA in pharmaceutical dosage forms and biological fluids including spectrofluorometric, micellar electrokinetic chromatography, differential pulse voltammetry, HPLC, LC/MS/MS and spectrophotometric [3-6]. However, the most widely used and applied methods are those based on spectrophotometry due to its sensitivity, specificity and simplicity. Spectrophotometric methods are based on three principles: (i) diazotization of an organic amine and subsequent coupling with reagents such as 8-hydroxiquinolin, -naphthol, resorcinol, phloroglycinol, diphenylamineto form an azo dye [7-9], (ii) nucleophilic reaction of benzofurazan and benzofurocxan reagents (4-chloro- 5.7 dinitrobenzofurazan, 7-chloro-4,6 dinitrobenzofurocxan, 5.7-dichloro-4, 6-dinitro- benzofuroxan) with mesalazin in polar media forms a stable product with a strong red color [2], and (iii) Schiff bases are formed by a condensation reaction between mesalazine and an aldehyde or ketone such as vanillin, cinnamaldehyde, p-dimethyl- aminobenzaldehyde [6, 10]. However, the disadvantages of using these methods are that the reaction is often narrow linearity range, requiring heating or extraction, long time for the reaction to complete, use of non- aqueous systems, low stability of the colored product formed. Spectrophotometric methods based on diazotization and coupling principle are sensitive and specific. N-(1-Naphthyl)ethylenediamine dihydrochloride (NEDA) is a simple diamine reported as a coupling agent in spectrophotometric analysis of thiols, aromatic amines, sulfonamides, aminophenols, dinitroanilines, and chloroanilines. It is widely used for the determination of drugs and pharmaceutical containing free primary aromatic amino group. The objective of the investigation reported in this paper was to evaluate a simple spectrophotometric method for the determination of mesalazine. Based on the diazotization of 5-ASA and coupling with N-(1-naphthyl)ethylenediamine VJC, 54(4) 2016 Nguyen Trung Dung, et al 510 dihydrochloride (NEDA) reagent and applying the method to the determination of 5-ASA in pharmaceutical preparation. Figure 1: Metabolism of mesalazine and its prodrugs in in humans body [2] 2. EXPERIMENTAL 2.1.Chemicals and equipment A Biochrom Model SP-60 double beam, UV- VIS spectrophotometer (Biochrom Ltd., UK) with 1.0 cm matched quartz cells was used for absorbance measurements. Mesalazine (Sigma-Aldrich, Germany, certified to be 99.0 %) and N-(1-naphthyl) ethylenediamine dihydrochloride (NEDA) (Maya - R, China, certified to be 99 %) were used. All other chemicals and solvents used were of analytical reagent grade. Commercial samples of tablets namely Pentasa (Ferring, Switerland) and Sunmesacol (Sunpharma, India) containing 500 and 400 mg of mesalamine, respectively were purchased from local pharmacy market and employed in the study. 2.2. Standard solutions A stock solution of mesalazine (1 mg/ ml) in absolute ethanol. The working standard solution of mesalazine containing 50 µg/ml was prepared by dilution. Hydrochloric acid solution, 1 N. This solution was prepared by diluting 8.5 ml of the concentrated acid to 100 ml with distilled water. Sodium nitrite solution, 1 %. This solution was prepared by dissoslving 1 g of sodium nitrite in 100 ml distilled water in a volumetric flask. Sulphamic acid solutions, 3 %. This solution was prepared by dissolving of 3 g of sulphamic acid in 100 ml distilled water. NEDA solution, 0.1 %. This solution was prepared by dissolving 0.1 g of NEDA in distilled water in a 100 ml volumetric flask. Pentasa and Sunmesacol tablets solution 1 mg/ml. Weigh and mix the contents of ten tablets (each one contains 500 mg 5-ASA for Pentasa and 400 mg 5-ASA for Sunmesacol), an accurately weighed amount of powder equivalent to 0.1 g 5- ASA was dissolved in 10 ml absolute ethanol and 30 ml distilled water, after filtration of the solution, the volume of filtrate was completed to 100 ml with distilled water in a volumetric flask. The working Pentasa and Sunmesacol tablet solution of mesalazine containing 50 µg/ml was prepared by dilution. 2.3. Procedure and calibration graph Into a series of 10 mL volumetric flasks, volumes of 5-ASA working standard solution equivalent to 0.5-40 μg/ml were transferred. To each flask, 2.0 ml of hydrochloric acid (1 N) and 0.5 ml of sodium nitrite (0.3 % w/v) were added and a reaction time of 5 minutes at 0-5 °C was given for completion of the reaction. Next, 0.5 ml of sulphamic acid (3 % w/v) was added to each flask with gentle shaking and after 3 minutes, 5 ml of NEDA reagent (0.1 %w/v) was added, and kept for 5 minutes. Finally the volume in each flask was brought up to the 10 ml mark with distilled water. The absorbances of violet-colored chromogen were measured at 552 nm against the reagent blank and and a calibration graph was constructed. The colored chromogen was stable for twenty four hours. VJC, 54(4) 2016 Development of spectrophotometric method for 511 2.4. Statistical analysis The limit of detection (LOD) and quantification (LOQ) of the method are given by and respectively, relative standard deviation (RSD (%))= ; where SD is the standard deviation, b is the slope of the calibration curve equation, " is the average value of the measurement. The % recovery of the added pure drug was calculated as: % Recovery = [(Ct–Cs)/Ca] x 100 where Ct is the total drug concentration measured after standard addition; Cs, drug concentration in the formulation sample; Ca, drug concentration added to the formulation Calculation and processing of data were done using the programs Origin Pro 8.0 and Statistica 7 (US). 3. RESULTS AND DISCUSSION 3.1. Principles of the method The method is based on diazotization of mesalamine with nitrous acid, to form diazotized mesalamine (1). The residual nitrite (as nitrous acid) which was undesirable due to its side reaction, such as, nitrosation of coupling agent, was removed by sulphamicacid (2), followed by its coupling with NEDA reagent to form a violet colored chromogen (3) with maximum absorption at 552 nm; it obeyed the Beer’s law in the concentration range of 0.4-40 μg/ml. The reaction mechanism is shown in figure 2. Figure 2: The reaction mechanism 3.2. Study of the optimum reaction conditions The various parameters affecting the colour intensity of the dye have been studied and optimum conditions are selected. 3.2.1. Choice of coupling agent Different coupling agents are used for the reaction with diazotized mesalazine. The results in table 1 indicated that NEDA gave the highest intensity with a good colour contrast for coloured product. Table 1: Selection of coupling agent Reagents 0.1 % Absorbance λmax(nm) Δλ * nm α-napthylamine 0.148 381 83 8-hydroxyl quinoline 0.16 407 114 Phenol 0.186 318 40.5 Diphenylamine 0.28 341 44.5 Aniline 0.38 330 70 NEDA 0.47 552 82 *Δλ= colour contrast = λmaxS - λmaxB, where S = The dye, B = Blank. 3.2.2. Effect of acids on the diazotization The effect of the amount of different acid (week and strong) for the diazotization of 5-ASA, has been investigated. The results indicated that 2.0 ml of 1 N HCl produces the highest intensity for the dye, so it has been selected in the subsequent experiments (figure 3). Figure 3: Effect of diazotization acid on absorbance 3.2.3. Effect of nitrite amount and time The colour reached maximum intensity when using 0.5 ml of 1 % (w/v) sodium nitrite solution VJC, 54(4) 2016 Nguyen Trung Dung, et al 512 within 5 minutes reaction time (table 2). It seems that diazotization of 5-ASA was fast. 3.2.4. Effect of sulphamic acid amount and time The presence of unreacted nitrite was undesirable in diazotization reaction. Therefore, it should be removed by sulphamic acid which quickly reacts with nitrite. The results indicated that 0.5 ml of 3 % sulphamic acid solution with 3 minutes standing time was considered to be the most suitable (table 3) and therefore was selected subsequently. Table 2: Effect of nitrite amount and time on absorbance ml of 1% (w/v) NaNO2 solution Absorbance/minute standing time 0 1 2 3 5 7 10 0 0.012 0.093 0.029 0.018 0.154 0.112 0.201 0.5 0.137 0.303 0.361 0.411 0.433 0.331 0.303 1.0 0.213 0.28 0.38 0.332 0.388 0.303 0.287 1.5 0.356 0.366 0.337 0.319 0.374 0.211 0.32 2.0 0.305 0.350 0.349 0.376 0.406 0.304 0.271 2.5 0.056 0.065 0.048 0.04 0.179 0.14 0.237 Table 3: Effect of sulphamic acid and time on absorbance ml of 3% (w/v) sulphamic acid solution Absorbance/minute standing time 0 1 2 3 5 7 10 0 0.002 0.017 0.019 0.02 0.013 0.015 0.011 0.1 0.003 0.029 0.034 0.063 0.042 0.021 0.014 0.3 0.006 0.023 0.055 0.266 0.063 0.044 0.023 0.5 0.024 0.148 0.274 0.401 0.320 0.291 0.029 0.7 0.054 0.146 0.231 0.265 0.177 0.151 0.019 1.0 0.044 0.185 0.235 0.305 0.271 0.203 0.188 1.5 0.057 0.164 0.244 0.294 0.221 0.162 0.114 2.0 0.062 0.102 0.199 0.274 0.207 0.192 0.142 3.2.5. Effect of NEDA amount The effect of NEDA amount on the colour intensity of the dye has been studied. From the result it can be observed that 5 ml of 0.1 % NEDA was the most suitable amount which gives the highest value of absorbance for the azo-dye formed (table 4). Table 4: The effect of NEDA amount Amount of 0.1 % NEDA (ml) Absorbance / μg of 5-ASA 5 10 15 0.5 0.031 0.150 0.336 1.0 0.081 0.279 0.668 2.0 0.315 0.506 1.089 3.0 0.364 0.685 1.694 4.0 0.475 0.913 1.87 5.0 0.518 1.006 2.301 6.0 0.500 0.966 2.151 7.0 0.477 0.853 1.941 3.2.6. Final absorption spectra When 5-ASA was treated according to the recommended procedure, the absorption spectrum shows a maximum absorption at 552 nm. characteristic of the violet dye. The reagent blank shows no absorption at this wavelength (Fig. 4). Figure 4: Absorption spectra of 5-ASA-NEDA and blank VJC, 54(4) 2016 Development of spectrophotometric method for 513 3.3. Comparison with other spectrophotometric methods The proposed method compares favorably with other reported methods. As shown in table 5 the proposed method is more sensitive than other methods in that it needs no heating and the product is stable for a longer time. 3.4. Interference The extent of interference by some excipients magnesium stearate, glucose, lactose, glycine and starch which are often found in pharmaceutical preparations was studied by measuring the absorbance of solutions containing 5 µg/ml of mesalamine and 500, 1000 and 2000 µg/ml of excipients in the final volume of 10 mL. It was found that these excipients do not interfere in the present method. The range of recovery is between 95.4 and 102.3 %. We consider that this variation is acceptable. Table 5: Comparison of the proposed method with other spectrophotometric methods Parameters Proposed method [9] [10] λmax (nm) 5525 510 440 Reagents NEDA α-Napthol PDAB Beer´s limit (µg/mL) 0.5-40 1-15 50-500 Molar absorptivity (L.mol -1 .cm -1 ) 1.50×10 4 1.48×10 4 3.74×10 2 Sandell’s sensitivity (µg/cm2) 0.02633 0.01030 0.415 Stability of colored products (h) 24 3 5 Regression equation (Y = bx + a) Slope (b) 0.0851 0.5578 0.0237 Intercept (a) 0.0985 0.0152 0.0041 Correlation coefficient (R 2 ) 0.999 0.9975 0.9980 Relative standard deviation (%) 0.615 0.9216 0.00436 LOD (µg/mL) 0.113 0.0059 0.699 LOQ (µg/mL) 0.342 0.0179 1.84 Table 6: Effect of interferences Foreign compounds Recovery (%) 500 1000 2000 Magnesium stearate 97.0 100.1 100.4 Glucose 99.2 98.4 96.1 Lactose 102.3 102.0 101.9 Glycine 97.2 95.4 100.7 Starch 97.2 100.3 95.5 3.5. Application of the method The results of analysis of marketed formulation are shown in table 7. The relative standard deviation values are below 2 % indicating the precision of the method. The validations of the proposed methods were further confirmed by recovery studies. The % recovery varies from 98.52±0.712 to 99.68±0.692 indicated high accuracy of methods. The high % recovery value indicates non interference from excipients used in formulations. 4. CONCLUSION The proposed methods are found to be simple, sensitive, selective, accurate, precise and economical and can be used in the determination of mesalamine in pharmaceutical preparation. Table 7: Results of analysis of marketed tablets Brand name of tablet dosage form Labeled amount (mg/tablet) Amount found by proposed methods Recovery (%) RSD (%) Pentasa 500 498.45 99.68±0.692 0.694 Sunmesacol 400 394.09 98.52±0.712 0.722 VJC, 54(4) 2016 Nguyen Trung Dung, et al 514 REFERENCES 1. P. G. Javier, G. Fernando, M. José, M. P. José. 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Development and Validation of Spectrophotometric Methods for the Estimation of Mesalamine in Tablet Dosage Forms, J. Young Pharm, 2(3), 284-288 (2010). Corresponding author: Nguyen Trung Dung Faculty of Physics an Chemical Engineering Le Quy Don Technical University 236 Hoang Quoc Viet St, Cau Giay Dist, Hanoi E-mail: nguyentrungdung1980@gmail.com; Tel.: 0915191424.