Spectrophotometric Dertermination of Mesalazine by 8-Hydroxyquinoline and N-(1-naphthyl)ethylenediamine dihydrochloride Reagents in Bulk and Capsule Dosage Forms

Two simple and sensitive visible spectophotometric methods have been developed for the quantitative determination of mesalazine (MEZ) in bulk drug and pharmaceutical preparation (capsules). The proposed methods are based on oxidative coupling reaction of mesalazine with 8-hydroxyquinoline (method A) and N-(1-naphthyl)ethylendiamine (NNED) (method B) in the presence of sodium periodate as oxidizing agent in alkaline medium to form coloured products, exhibiting maximum absorptions at 644 nm and 543 nm, respectively. Beers law was obeyed in the concentrations range of 10-300 and 10-180 μg /25ml with molar absorptivity of 1.7223×10 and 0.6545×10 l.molcm, respectively. The relative error ranged between -0.4% to +0.16% and 0.9% to 0.31% with relative standard deviation of ±1.31% to ±0.39% and ±0.88% to ±0.32% for methods A and B, respectively. The optimum conditions for full colour development are described and the proposed methods were applied successfully to the assay of MEZ in pharmaceutical preparation (capsules).


INTRODUCTION
Mesalazine (MEZ) is chemically known as 5-amino-2-hydroxybenzoic acid, it is a white to pinkish, crystalline powder, slightly soluble in cold water and alcohol, more soluble in hot water, soluble in hydrochloric acid.It is an anti-inflammatory drug used to treat and also maintain the remission of mild to moderate ulcerative colitis or Crohn ' s (Madhavi et al., 2011 ;Singh et al., 2010).
Various methods have been reported for the determinations of MEZ as pure and in pharmaceutical preparations, these include: High performance liquid chromatography (HPLC) (Darak et al., 2012) and (Nobilis et al., 2006), reversed-phase-HPLC (RP-HPLC) (Majji et al., 2008) have been also used in the determination of MEZ in pharmaceutical preparations and blood plasma .
A Square wave voltammetric method (SWV) was applied to the determination of MEZ in pharmaceuticals by using pencil graphite electrodes (Uliana et al., 2010).
A Fluorimetric method has been employed for the determination of MEZ in blood serum, the fluorescence intensities were measured at 480 nm with excitation at 340 nm (Zadeh and Kolansal, 2012).
UV-spectophotometric methods were developed for the determination of MEZ in pure form and pharmaceuticals based on the measurement of absorbance at 210 nm in methanol and 303 nm in 0.5N HCl, respectively (Singh et al., 2010 ;Moharana et al., 2011).
The present work describes two simple and accurate spectrophotometric methods for the determination of MEZ in pure and pharmaceutical preparation (capsules).

EXPERIMENTAL Instruments
Spectrophotometric measurements were carried out using Shimadzu UV-160 and UV-Visible spectrophotometer CECIL-CE 1021 digital single beam using 1-cm silica cells.pH meter type Philips PW 9420 was used for pH reading.

Reagents
All chemicals used in this investigation were analytical grade reagents.Working MEZ solution, 100 µg/ml.A 0.01g of MEZ (Fluka) was dissolved in 10 ml of absolute ethanol to increase solubility and diluted to 100 ml with distilled water in a volumetric flask.N-(1-naphthyl)ethylenediaminedihydrochloride (N-NED) solution, 0.005M.This solution was prepared by dissolving 0.1295g of N-NED (Fluka) in 100 ml distilled water.8-hydroxyquinoline solution, 0.01M.This solution was prepared by dissolving 0.145g of 8-hydroxyquinoline (Fluka) in absolute ethanol in a 100 ml volumetric flask.Sodium periodate solution, 0.015M.This solution was prepared by dissolving 0.321 g of sodium periodate in distilled water and made up to 100 ml in a volumetric flask with the same solvent.Sodium hydroxide solution, 1N.This solution was prepared by appropriate dilution of the concentrated volumetric (Fluka) solution with distilled water to 1000 ml in a volumetric flask and then transferred to a plastic bottle.Mesacol (capsules) solution, 100 µg /ml MEZ solution from Mesacol Capsules.This solution was prepared by weighing and mixing the contents of ten capsules (each one contains 400 mg MEZ), an accurately weighed amount of powder equivalent to 0.01g MEZ 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.

Method-A
To a series of 25-ml volumetric flasks, 0.1-4 ml of MEZ solution (100 µg /ml ) were transferred, followed by the addition of 2 ml of 8-hydroxyquinoline (0.01 M), 0.5 ml of sodium periodate (0.015M) and 2 ml sodium hydroxide(1N).The solutions were left to stand for 10 minutes before the volumes were completed to the mark with distilled water.After 15 minutes, the absorbances were measured at 644 nm against the reagent blank.The calibration graph was linear over the concentration range of 10-300 µg MEZ /25 ml (0.4-12 ppm), a concentration above 300 µg /25 ml gave negative deviation from Beer ' s law (Fig. 1).The molar absorptivity is 1.7223×10 4 l.mol -1 cm -1 .Method-B To a series of 25 ml volumetric flasks, 0.1-2 ml of MEZ solution (100 µg /ml ) were transferred, followed by the addition of 0.5 ml of sodium periodate (0.015M), 1ml of N-NED (0.005 M) and 1ml of sodium hydroxide (1N).The solutions were left to stand for 15 minutes before the volumes were completed to the mark with distilled water.After 15 minutes the absorbances were measured at 543 nm against the reagent blank .The calibration graph was linear over the concentration range of 10-180 µg MEZ /25 ml (0.4-7.2 ppm), a concentration above 180 µg /25 ml gave negative deviation from Beer ' s law Fig.

RESULTS AND DISCUSSION
The effect of various parameters on the colour development of 100 µg of MEZ were considered in 25 ml final volume and the absorbance measurements were performed at 644 and 543 nm for the methods A and B, respectively.

Choice of oxidizing agent and its concentration
Different types of oxidizing agents were used for the purpose of producing intense coloured product and strong colour contrast [Table (1)].The effect of different volumes (0.3-2 ml) of NaIO 4 solution (0.015M) on the colour intensity has been studied, it was observed that 0.5 ml of NaIO 4 for both methods is the most suitable volume therefore it was chosen for the subsequent experiments.

Effect of 8-hydroxyquinoline and N-NED reagents concentration
The effect of 8-hydroxyquinoline and N-NED concentrations on the absorbance of the complex in method A and B, respectively were investigated.It was found that 2 ml of 8hydroxyquinoline (0.01M) and 1ml of N-NED (0.005M) gave maximum absorbance which were then used in subsequent experiments Table (2).

Choice of the base and its amount
The preliminary experiments have shown that MEZ gave an intense coloured dye with 8-hydroxyquinoline (method A) and N-NED (method B) in the presence of sodium periodate in alkaline medium, so that different bases are examined Table (3).The results shown in Table 3 indicated that NaOH gave the highest colour intensity of the product and a good colour contrast for both methods .The effect of different volumes (0.5-4 ml) of 1N NaOH solution on the colour intensity was studied then, a 2 ml and 1 ml of 1N NaOH with a final solution pH of 11.8 and 12.48 gave the highest intensity of the formed products for methods A and B, respectively.

Effect of time on oxidative coupling reaction
The time required for complete oxidation was tested in both methods.The results indicated that 10 minutes (method A) and 15 minutes (method B) were needed to give complete oxidation process before dilution with distilled water in both methods.

Effect of surfactant
The effect of different surfactants on the colour intensity were studied by using 1 ml of various types of surfactants.The results reveal that non of the surfactants give useful results from the analytical point of view.Therefore, it has been recommended to eliminate their use in the subsequent experiments in both methods [Table (4

Effect of the order of addition
The effect of the order of addition on the absorbance of the product was studied under the optimum experimental conditions [Table (5)].From the results above, order II for method A and order I for method B have been used for subsequent experiments due to the highest sensitivity.

Effect of temperature on the absorbance
The effect of temperature on the colour intensity of the resulting product was investigated.In practice, the high value of absorbance was obtained when the colour was developed at room temperature (25 C ° ±1), but when the volumetric flasks were placed in an ice-bath at (0 C °) or in a water-bath at (50 C °) a loss in colour intensity and stability were observed.It is therefore recommended that the colour reaction should be carried out at room temperature for both methods.

Development time and stability period
The effect of time development and stability period of the coloured complex was investigated under the optimum conditions of the reaction.The stability of the colour intensity was reached after about 15-20 minutes and the absorbance of the colour complex remained constant for at least one hour for both methods.[Table (6)].

Final absorption spectrum
When MEZ was treated according to the recommended procedure, the absorption spectrum showed a maximum absorption at 644 nm (method A) and 543 nm (method B) versus the blank (Fig. 3 and 4).(A) the complex against blank, (B) the complex against distilled water, (C) the blank against distilled water.

Accuracy and precision
To check the accuracy and precision of the calibration graph, MEZ was determined at three different concentrations.The results shown in Table ( 7), indicate that a satisfactory accuracy and precision could be obtained with the proposed methods.

The nature of the reaction product
Mole ratio method indicates that the coloured product has a composition of 1:1 MEZ to 8-hydroxyquinoline reagent at 644 nm (Fig. 5) and 2:1 MEZ to N-NED reagent at 543 nm (Fig. 6).

Fig. 6: Mole ratio's plot of MEZ-N-NED coloured product
Therefore, the probable reaction path might be written as follows :

Interferences
In order to asses the possible analytical application of the proposed methods, the effect of some foreign substances which often accompany the pharmaceutical preparations were studied by adding different amounts of these foreign substances to 100 µg MEZ /25 ml.It was found that the studied foreign species did not interfere in the present methods Table (8).

Analytical applications
The proposed methods were successfully applied to the determination of MEZ in its pharmaceutical preparation (capsules) as good recoveries were obtained Table (9).

Evaluation of the proposed methods
Because the standard method for the determination of MEZ involves potentiometric titration and according to difficulties of availability of using it, standard addition method was used in order to prove that the proposed methods can be used in the determination of MEZ in pharmaceutical preparation (capsules) Table ( 10

Comparison of Methods
Table (11) shows the comparison between the analytical variables from the present methods with that of a recent spectrophotometric method.

CONCLUSION
The proposed visible spectrophotometric methods for the estimation of MEZ are simple, sensitive, accurate and precise.Method A (used 8-hydroxyguinoline) was found to be more sensitive compared to method B (used N-NED) for the assay of MEZ and can be used for the routine quality control of the drug in bulk as well as in a pharmaceutical preparation (capsules).
Absorption spectrum of MEZ with N-NED at 543nm ) and Fig.(7)

Fig. 7 :
Fig.7: Calibration standard addition graphs for the determination of MEZ in capsulesfor both methods

Table 1 : Selection of oxidizing agent
The results in Table1indicated that NaIO 4 gave the highest intensity with a good colour contrast for coloured product in both methods A and B.

Table 4 : Effect of surfactant
max S   -λ max B

Table 5 : Effect of the order of addition
Assuming that: S = sample, R = reagent, B = base, O = oxidizing agent

Table 7 : Accuracy and precision of the proposed methods
* Average of five determinations

Table 9 : Application of methods
* Average of five determinations

Table 10 :The results of standard addition method
• Average of three determinations