Spectrophotometric Determination of Mesalazine in Pharmaceutical Preparations by Oxidative Coupling Reactions with m-Aminophenol and 2,6- Dihydroxybenzoic Acid

Tow simple, rapid and sensitive spectrophotometric methods for the determination of mesalazine in pharmaceutical preparations have been carried out. The proposed methods depend on oxidative coupling reaction of mesalazine with m-aminophenol in the existence of N-bromosuccinamide in alkaline medium (method A) and 2,6-dihydroxybenzoic acid in the existence of sodium metaperiodate in basic medium (method B) to produce colored products , show highest absorptions at 640 (nm) and 515 (nm), alternately. Beer’s law was consistent in concentrations extent of 1.25-30 and 0.5-12.5 (μg.mL -1 ) with molar absorptivity of 0.36×10 4 and 0.77×10 4 L.mol -1 .cm -1 , alternately. The limits of detection (LOD) were 0.0144 and 0.0829 μg.mL -1 , while limits of quantitation (LOQ) were 0.0483 and 0.2766 (μg.mL -1 ), alternately. The suggested methods have been applied successfully to the determination of mesalazine in its pharmaceutical preparations as tablets and suppositories.


Introduction:
Mesalazine (MZ), also known as mesalamine or 5-aminosalicylic acid (5-ASA), is an anti inflammatory drug used to treat inflammation of the digestive tract (Crohn's disease) and mild to moderate ulcerative colitis (1). The mechanism of action of MZ is unknown, but appears to be topical rather than systemic. Mesalazine acts as a scavenger of oxygen-derived free radicals, which are produced in greater numbers in patients with inflammatory bowel disease (2). Mesalazine is administered orally or rectally in the treatment of ulcerative colitis and Crohn's disease (3). The chemical structure of mesalazine as it is shown in Fig. 1 :

Figure 1. Chemical structure of mesalazine
Mesalazine consist of white to pinkish crystals, decomposed at about 280 °C, slightly soluble in cold water, alcohol more soluble in hot water and in hydrochloric acid (4) . Department of Chemistry, College of Science, Mosul University, Mosul, Iraq * Corresponding author: saad.hasani@yahoo.com A number of analytical methods have been reported for the assay of mesalazine in pharmaceutical dosage forms. These methods include different techniques such as: spectrophotometry (5-10), spectrofluorometry (11), high-performance liquid chromatography (12)(13)(14) and voltammetry (15,16). However these methods generally require expensive equipment, provision for use and personal skills , so it still seems to be that the spectrophotometric methods are more convenient than other techniques due to their inherent simplicity , high sensitivity , low cost and wide availability in most laboratories .To the best of our knowledge, the reagents m-aminophenol and 2,6-dihydroxybenzoic acid were not previously used in oxidative coupling reactions, so the aim of this project is to employ these reagents for the assay of mesalazine in its pharmaceutical preparations .

Materials and Methods: Apparatus
All spectrophotometric measurements were carried out on Jasco V-630 UV-Visible spectrophotometer with 1.0 cm matched glass cells, pH measurements were performed by pH meter type TRANS BP 3001.

Open Access
Reagents and chemicals All reagents and chemicals used were analytical grade .

Mesalazine solution, (100 µg/mL )
The solution was attended by dissolving 0.01 g of pure mesalazine (Aldrich) in 5 mL ethanol and 40 mL distilled water with gentle heating, then the volume was completed to the mark with distilled water . N-bromosucinamide solution, (0.1%) 0.1 g of pure substances (Reidel-Haen) was dissolved in enough amount of distilled water, then the volume was completed to 100 mL by distilled water in avolumetric flask . m-Aminophenol reagent solution, (0.1%) The solution was attended by dissolving 0.01g of m-aminophenol (Fluka) in enough amount of distilled water with gentle heating and the volume was completed to (100) mL by distilled water in a volumetric flask .

2,6-Dihydroxybenzoic acid solution, (0.1 %)
This solution was attended by dissolving 0.1 g of pure reagent (Fluka) in enough amount of distilled water with gentle heating and the volume was completed to (100) ml by distilled water in avolumetric flask .

Sodium hydroxide solution, (1M)
This solution was attended by the appropriate dilution of the concentrated volumetric (BDH) solution with distilled water and then transferred to a plastic bottle. Tablets (Pentasa and Awasalazine) solution, (100 µg.mL -1 ) The contents of ten tablets (each tablet contains 500 or 400 (mg) mesalazine as Pentasa or Awasalazine preperations) were finely grinded, mixed well and weighed accurately to a quantity equal to (0.01 g) of mesalazine was dissolved in (5 mL) ethanol and (40 ml) distilled water with gentle heating and after filtration of the solutions, the volumes were made up to (100 ml) by distilled water in a volumetric flask .

Suppositories (Asacol) solution, (100 µg.mL -1 )
The content of three Asacol suppositories(each one contains 500 mg of mesalazine) are mixed well, an accurately weighed equivalent to (0.01 g) was dissolved in (5 ml) ethanol and (40 ml) distilled water with gentle heating, then the solution filtrate using a filter paper and the volume was made up to (100 ml) by distilled water in a volumetric flask .

Recommended procedures (Method A)
Accurately measured volumes containing (25-600) µg of mesalaszine were transferred into a series of 20 ml calibrated flasks, followed by the addition of 1.0 ml of 0.1% N-bromosuccinamide and 0.5 ml of 0.1% m-aminophenol ,then adding 1.0 mL of 1.0 M sodium hydroxide and the volumes completed to the mark with distilled water and then measure the absorbance at 640 nm versus the reagent blank solution .

(Method B)
Accurately measured volumes containing 10-250 (µg) of mesalaszine were transferred into a series of 20 ml calibrated flasks, followed by the addition of 1.5 ml of 0.1% 2,6-dihydroxybenzoic acid, 1.5 ml of 0.1% sodium periodate and 1.5 mL of (1.0 M) sodium hydroxide and the volumes were completed to the mark with distilled water and then the absorbance was measured at 515 (nm) versus the reagent blank solution .

Results and Discussion:
The effect of different factors on the color evolution of (100 µg) of mesalazine was investigated in 20 ml total volume and the absorbance measurements were carried out at 640 and 515 nm for the methods A and B , alternately .

Effect of m-aminophenol and 2,6-dihydroxybenzoic acid reagents amounts
The effect of m-aminophenol and 2,6dihydroxybenzoic acid reagents amounts on the absorbance of the colored product in method A and B , alternately was studied . The results in Table 1 indicate that 0.5 mL of 0.1% m-aminophenol and 1.5 mL of 0.1% 2,6-dihydroxybenzoic acid give the highest absorbance which were chosen for the next experiments.

Effect of oxidizing agent amount
The effect of oxidizing agent amount on the absorbance of the formed dyes for both methods hase been investigated. The results noticed from Table 2 that 1.0 mL of 0.1% N-bromosuccinamide and 1.5 mL of 0.1% sodium metaperiodate were the ideal volumes, so they were select for the next experiments.

Effect of bases type and its quantities
The initial tests revealed that mesalazine produced colored dyes for two methods (A and B) just in alkaline medium, so the effect of different bases on the absorbance was investigated and the obtained results are illustrated in Table 3. They indicate that sodium hydroxide forms the intense dye for both methods compared with the other bases, so different amounts of sodium hydroxide were added and the obtained results reveal that 1.0 mL and 1.5 mL of 1M of NaOH are the optimum volumes for method A and B, respectively . The effect of sequence of addition The effect of sequence of addition on the absorbance of colored products was investigated. The results listed in Table 4 indicate that order (I) for method A and order (II) for method B have been chosen for the next experiments because they gave high sensitivity .

Relationship between temperature and absorbance
The role of temperature on the absorbance of the formed dyes was studied by applying the procedure for methods A and B under optimum conditions at three different temperatures .The obtained results listed in Table 5 reveal that room temperature (20±2 °C) gave high value of absorbance, while conducting the reaction at (0 °C) or in (40 °C) reduced the sensitivity of the methods, therefore it is better to do the reaction in room temperature due to its better sensitivity and simplicity.

Effect of surfactants
The results obtained from the investigation of three types of surfactants (CTAB, SDS and Triton X-100) on the sensitivity of proposed method A and B revealed that there is no improvement in the intensity of the formed dyes, so it is unfavorable to use it .

Effect of time on color development
The effect of time development and stability period of the colored product was investigated under the optimum conditions. From Table 6, it is noticed that the formed dyes in method( A and B) reached maximum absorbance immediately after the addition of the base and stayed stable about 2 hrs. in which many measurements can be done .

calibration graphs for proposed methods
According to the obtained optimum conditions the calibration curves for method (A) and (B) Fig.2, were linear over the concentrations extent (1.25-30 µg.mL -1 ), and (0.5-12.5 µg.mL -1 ), respectively .The apparent molar absorption referred to mesalazine has been found to be 0.36×10 4

.Calibration graphs for MZ determination (method A & B)
The final of absorption spectra Using the recommended procedures in both methods (A and B), the formed dyes show a final absorption spectra with maximum absorbance at 640 nm (method A) and at 515 nm (method B), against the blank solutions as shown in Fig. 3 and 4.

Nature of the formed dyes
To estimate the reaction ratio of MZ to m-aminophenol and 2,6-dihydroxybenzoic acid reagents, Job's method was used, which indicates that the formed dyes in both methods have a composition ratio 1:1 as shown in Fig. 5 and 6.

A Effect of additives species
The effect of some added compounds which are often found in the pharmaceutical formulations was examined by adding various quantity of this additives to (100 µg) of mesalazine using the recommended procedures. The results in Table 7 indicate that none of added compounds can introduce significant interference.

Application of the proposed methods
To test the applicability of the proposed methods, they the determination of mesalazine in its pharmaceutical preparations (tablets and suppositories) has been applied. The results shown in Table 8 indicate that the proposed methods have good accuracy, precision and recovery.

Evaluation of the proposed methods
The efficiency of the proposed methods was checked by calculating the t-test for the present methods compared with the standard method of British pharmacopeia (18) for 95% confidence level with eight degree of freedom. The results in Table 9 indicate that the t-values were less than the critical value (2.306), which mean there are no real differences between the present methods and standard method for mesalazine determination.

Present methods compare
The proposed methods compared with other spectrophotometric methods, are illustrated in Table 10. Method (A) has a wide scale while method (B) has an acceptable range of determination and sensitivity compared with a recent spectrophotometric method.

Conclusion:
A simple, rapid and sensitive spectrophotometric methods are described for the assay of mesalazine in its pharmaceutical formulations as tablets and suppositories .The suggested methods depend on oxidative coupling reactions with m-aminophenol in the presence of N-bromosuccniamide as an oxidant agent in basic medium (method A) and with 2,6-dihydroxybezoic acid in the presence of sodium metaperiodate as oxidant agent in basic medium (method B) to form colored dyes were stable for at least 2 hrs at room temperature. The present methods do not need to control the temperature or extraction process.