Spectrophotometric Assay of Mesalazine in Pharmaceutical Preparations Via Oxidative coupling reaction with o-cresol and sodium metaperiodate

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Introduction
Mesalazine known as mesalamine as well. It is known chemically as 5-aminosalicylic acid ( Figure  1. It is used for the treatment of inflammatory browel disease, including ulcerative colitis and Crohn's disease [1].
The current work aims to create and validate a new and simple spectrophotometric method for the detection of mesalazine in bulk and pharmaceutical dosages. The proposed method is based on the oxidative coupling reaction between mesalazine, o-cresol, and sodium metaperiodate in an alkaline medium to form a stable dye with blue color.

Experimental Apparatus
A double beam spectrophotometer Shimadzu model UV-160A with 1.0 -cm matched quartz cells was used to carry out all spectral and absorbance measurements.

Reagent
All chemicals used were of analytical grade and used without further purification.
Mesalazine was provided from SDI-Iraq, o-cresol, sodium metaperiodate and sodium hydroxide were obtained from Fluka company.

Standard solution of Drug ( mesalazine 100 µg/ml):
was prepared by dissolving 0.0100 g of pure drug in a minimum amount of ethanol and the mixture diluted to the mark with distilled water in a 100 ml volumetric Flask.

o-Cresol solution (0.01 M)
The reagent was prepared by dissolving 0.1081 g of o-cresol with the smallest volume of ethanol and diluted up to the mark (100 ml) with distilled water. In addition, Sodium metaperiodate (0.015M) was prepared by dissolving 0.8021g in 250 ml with DW.

Sodium hydroxide solution (0.1 M)
Alkaline er solution was prepared by dissolving 0.400 g in 100 ml of distilled water.

Recommended procedure:
To a series of 25 ml volumetric flasks, 2.5 ml of 0.01M of the o-cresol solution was added followed by the addition of 1 ml of 0.015 M sodium metaperiodate solution and 5 ml of 0.1 M sodium hydroxide solution. Aliquots of standard solution (10-300 µg) were transferred to the flasks respectively. The contents were diluted up to the mark with DW and left for 10 min. After that, the absorbance was measured at 645 nm contrary to blank. A calibration curve was drawn and the regression equation was calculated ( Figure 2).

Procedure for pharmaceutical preparations:
Tablets: Ten tablets of mesacol (enteric-coated tablets) were weighted and crushed into a powder. An accurately weighed quantity equivalent to 400 mg of the pure drug (mesalazine) was dissolved in 10 ml of ethanol and completed to 100 ml with DW. The resulting solution was shacked well and filtered. A solution of 100 µg/ml of the drug was prepared by appropriate dilution and the measurement was carried out as described earlier under the recommended procedure.

Capsules:
The contents of ten mesacol capsules (extended-release capsules) were weighted, powdered, and mixed well. A weighted quantity equivalent to 400mg of pure mesalazine was dissolved in 10 ml ethanol and 0.2 M sodium hydroxide (5:5) to increase the solubility. After that, transferred into 100 ml and completed up to the mark with DW. The final solution was filtered and treated as mentioned previously.

Characteristic of Absorption Spectral
A blue indophenol dye has been formed when o-cresol react with mesalazine in an alkaline medium in the presence of sodium metaperiodate. The resulting product has maximum absorption at 645 nm as shown in Figure 3. However, the reagent blank has no absorbance at the same wavelength.

Reaction conditions:
In order to improve the suggested method, the effect of some experimental variables was considered. The optimization was done using a sample solution of mesalazine (4µg/ml) at 645 nm.

Effect of pH:
It was observed that the blue dye product formed only in an alkaline medium, therefore, the effect of different alkaline solutions (pH 9-12) and sodium hydroxide (pH > 12) were studied. It was confirmed that sodium hydroxide gave the best and highest color intensity of the dye product for mesalazine (Table 1) and the optimum amount of this base was found to be 5-7ml and 5ml which was used in the subsequent experiments ( Figure 4).  Figure 4: Effect of the amount of sodium hydroxide.

Effect of o-cresol concentration
The influence of the volume (1-5 ml) of 0.01M of the o-cresol solution was examined. The obtained results showed that 2.5ml of o-cresol gave a higher color intensity of the dye product and was recommended for all measurements ( Figure 5).

Effect of oxidizing agent:
Different oxidizing agents were tested such as N-bromosuccinimide, ferric chloride, potassium hexacyanoferrate(III), sodium metaperiodate, and sodium nitroprusside. The obtained results show in Figure6 that sodium metaperiodate gave a stable blue color dye product with maximum intensity compared to other oxidizing agents. It was found that 1.0ml of 0.015M sodium metaperiodate was sufficient to get the highest color intensity (Figure 7).

Effect of temperature
After optimization of chemical variables, the effect of temperature on the reaction of mesalazine and o-cresol in the presence of sodium metaperiodate in the range of 0-50 C was studied. Practically, high absorbance was achieved at room temperature (23±2 C) as shown in Table 2.  Temp.

( C )
Absorbance/ min standing time The color intensity of the dye formed reached a maximum after 10 min and was stable for at least 4hr at room temperature.

Order of addition of the reactants:
To achieve high color intensity, the order of addition of reactant should be followed serial No.III of as shown in Table 3, otherwise a loss in product color intensity was experimentally detected.

Linearity of the method and sensitivity:
Under the optimum conditions, a linear relationship was reached between color intensity and concentration of mesalazine in the range 0.4-12µg/ml. The correlation coefficient, intercept, and slope for the calibration data was calculated in Table 4.
In addition, sensitivity parameters such as Sandell index and molar absorptivity, Limit of detection (LOD), and Limit of quantitation (LOQ) are calculated as presented in Table 4 [26].

Precision and accuracy:
The intraday precision and accuracy of the proposed method were examined by carrying out six replicates determination at a fixed concentration of mesalazine (within Beer's law range). The RSD (less than 3.5%) and R 2 (101.38%) were found to be in the acceptable range (Table 5)

Nature and stability constant of the dye product
Job's method [27] under optimized conditions was applied to calculate reaction stoichiometry. The obtained results showed a 1:1 mesalazine to o-cresol at 645 nm ( Figure 8). As a result, the formation of blue indophenol dye probably occurs as suggested in scheme 1 and as suggested previously [28,29]. The average stability constant [30] of the formed dye was 1.37x10 5 1.mol -1 which indicates the high stability of the product.

Interference
To evaluate the selectivity of the suggested method, the effect of common excipients for instance glucose, lactose, sucrose, starch, talc, acacia, magnesium stearate, and sodium chloride was tested by analyzing synthetic sample solutions containing 4 µg/ml of mesalazine and excess amount (10-20-fold excess) of each excipient. The results are listed in Table 6 and show no interference caused by the presence of these excipients.

Applications:
The recommended method was effectively applied for the determination of mesalazine in its dosage forms. The obtained results were compared with certified values (Table 7) and statistically to those obtained by the official method [4] utilizing t-and F-tests at 95% confidence level ( Table 8). The results indicated that there was no significant difference between the suggested and official methods in terms of accuracy and precision (Table 8).  Tabulated t-and F-testes at 95% confidence level are 2.31 and 6.39 respectively.

Conclusion:
The proposed method is economic, accurate, simple, and reproducible and can be used as routine analysis of mesalazine in bulk form and to analyze the pharmaceutical preparations (tablets and capsules).