Spectrophotometric Determination of Mesalazine by Diazotisation-Coupling Method with Resorcinol

A simple spectrophotoemtric method for the determination of mesalazine [5aminosalicylic acid; (5-ASA)] in aqueous solution is achieved. The method is based on the reaction of mesalazine, with excess nitrite, in an acidic medium, to produce the corresponding diazonium salt. After the removal of residual nitrite with sulphamic acid, the diazonium salt is coupled with resorcinol reagent in basic medium to produce, an intense orange coloured water-soluble and stable azo-dye which exhibits maximum absorption at 471nm. Beer’s law is obeyed in the concentration range of 10-300μg of mesalazine in a final volume of 25 ml i.e., 0.4-12 ppm with a molar absorptivity of 2.9480×10 l.mol.cm and Sandell sensitivity index of 0.0051μg.cm, a relative error of -0.96 to -0.23% and relative standard deviation of ±1.05 to ±0.37% depending on the concentration level. The proposed method has been applied successfully to determine mesalazine in pharmaceutical preparation (capsules). ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ ــــــــــــــــــــــــــــــــــ ــــ ا ةقيرط مادختساب نيزلاازيملل يفيطلا ريدقتلا لأ لونيسروسيرلا عم نارتقلااو ةتوز صخلملا نيزلاازيملا ريدقتل ةطيسب ةيفيط ةقيرط ثحبلا نمضتي ] 5 ا ضماح ونيمأ ،كليسلاسل (5-ASA) [ نارتقلااو ةتوزلأا ةقيرطب يئاملا طسولا يف . لاازيملا ةلعافم ىلع ةقيرطلا دمتعت نيز تيرتنلا نم ةدايز عم مث ،كيمافلسلا ضماح مادختساب ضئافلا تيرتنلا ةلازإو لباقملا موينوزايادلا حلم نيوكتل يضماح طسو يف ير فشاكلا عم توزؤملا نيزلاازيملا نارتقا متي تاذ ةـيوزآ ةغبـص يطعيل يدعاق طسو يف لونيسروس يجوـملا لوطلا دنع صاصتما ةدش ىلعا يطعتو ءاملا يف ةبئاذو ةرقتسم يلاقترب نول 471 رتيموناـن . نم زيكرتلا ىدم يف ريب نوناق دودح تناك 10 ىلا 300 يئاهن مجح يف نيزلاازيم مارغوركيام 25 رتللم يأ 0.4 12 ءزج / صاصتملاا تناكو نويلم ةجتانلا ةغبصلل ةيرلاوملا ةي 2.9480 × 10 رتل . لوم 1 . مس 1 ةيساسحلل لدناس ةللادو 0.0051 مارغوركيام . مس 2 . نم يبسنلا أطخلا 0.96 ىلا 0.23 % فارحنلااو


INTRODUCTION
Mesalazine [5-Amino salicylic acid; (5-ASA)] is an agent widely used in the treatment of inflammatory bowel disease (IBDs), is metabolized in organism to the principal biotransformation product, N-acetyl-5-ASA, is a polar compound and besides it exhibits amphoteric properties (Liu et al., 1995 andNobilis et al., 2006).
Different methods have been reported for the determination mesalazine including: liquid chromatographic technique which was used to determine 5-ASA and its metabolite N-acetyl-5-aminosalicylic acid in plasma and urine by using a spectrofluoimetric detector, excitation at 311nm.The lower limit of detection was 20 ng/ml and a relative standard deviation was below 6.7% (Bystrowska et al., 2000).
High performance liquid chromatography (HPLC) method was employed to measure 5-ASA and its metabolities in blood plasma.Chromatographic analysis were performed on a 250-4mm column with UV photodiode-array and fluorescence detectors (Nobilis et al., 2006).
A micellar electrokinetic chromatographic (MEKC) has been used for the estimation of mesalazine and its major impurities, the method used capillary chromatography column select fused-silica with a buffer solution (pH 10.20), methanol, sodium dodecyl sulfate (SDS), tetrabutylammonium bromide (TBAB) as a mobile phase.(Gotti et al., 2001).
Mesalazine was determined by three different methods: the first method (HPLC) was carried out with a C 18 column and a mobile phase was constituted of 30 mmol/l monobasic phosphate buffer (pH 7.0) and methanol (70:30; v/v), with 25% tetrabutylammonium hydrogen sulphate used ultraviolet detection at 254 nm, the second method used 1,1-diphenyl-2-picrylhydrazyl radicals (DPPH) at 517nm and using 100 mmol/l acetate buffer, pH 5.5, ethanol and 250 µmol/l ethanolic solution of DPPH and the third method nitrosation was accomplished using a platinum electrode and standard 0.1 mol/l sodium nitrite as titrant solution, the experimental recoveries were between 72.5 and 99.9%.All proposed methods can be used for the reliable quantitation of 5-ASA in pharmaceutical dosage forms (Rafael et al., 2007).
A differential pulse voltametry method was employed to determine 5-ASA in tablet using a glassy carbon electrode in Britton-Robinson buffer (pH 1.81), the peak current gave a linear relationship in the concentration range 1×10 -4 and 2×10 -6 molarity, the recovery was 101.23% with a relative standard deviation of 1.35% (Nigovic and Imunic, 2003).
A spectrophotometric method for the determination of microgram amounts (0.16-8µg/ml) of mesalazine based on the oxidative coupling with 2,6-xylenol in the presence of sodium metaperiodate in alkaline medium to form a blue indophenol dye which has maximum absorption at 610 nm with a molar absorptivity is 13316l.mol - .cm - (Al-Fakhry, 2006).
Another spectrophotometric method was applied for the determination of phenols based on a multicommuted flow system.It was based on oxidative coupling of phenolic .compounds with 4-amino-antipyrine in alkaline medium containing potassium hexacyanan ferrate (III).The detection limit was 1µg/l phenol.(Lupetti et al., 2004).
A colouremetric method has been developed for the determination of 5-ASA in urine and feces using Bratton-Marshall reaction to form a violet product with absorption at 560 nm.Beer's law was obeyed in the concentration range 0-70µg/ml (Pieniaszek and Bates, 1975).
Also, mesalazine has been determined by galvanostatic coulometric method in pharmaceutical preparations using reactions of electrogenerated bromide and chlorine with mesalazine.The end-point of coulometric titration was determined aperometrically with two polarized platinum electrodes.Procedures for the galvanostatic coulometric determination of 2.4 to 19.2 µg/ml with relative standard deviation varied from 1 to 5% (Abdullin et al., 2002).
The objective of the investigation reported in this paper is to introduce spectrophotometric method for the determination of 5-ASA.Based on the diazotization of 5-ASA and coupling with resorcinol reagent and applying the method to the determination of 5-ASA in pharmaceutical preparation (capsules).

EXPERIMENTAL
Spectral absorbance measurements are carried out on double beam spectrophotometric Shimadzu (UV-160A) and UV-visible spectrophoto-meter CECIL-CE 1021 digital single beam using 1 cm silica cells.

Reagents
All chemicals used are of the highest purity available.
Working mesalazine (5-ASA) solution, 50µg/ml.A 0.01g of mesalazine supplied by (Fluka) is dissolved in 10 ml distilled water, and the volume is completed to 200 ml in a volumetric flask.
Hydrochloric acid solution, 1N.This solution is prepared by diluting 8.5 ml of the concentrated acid to 100 ml with distilled water.
Sodium nitrite solution, 1%.This solution is prepared by dissolving 1g of sodium nitrite in 100 ml distilled water in a volumetric flask.
Resorcinol solution, 0.1%.This solution is prepared by dissolving 0.1g of resorcinol in distilled water in a 100 ml volumetric flask.
Sodium hydroxide solution, 1N.This solution is prepared by appropriate dilution of the concentrated (Fluka) solution with distilled water and then transferred to a plastic bottle.
Mesacol capsules solution 50µg/ml.Weight and mix the contents of ten capsules (each one contains 400 mg mesalazine), an accurately weighed amount of powder (0.0111g) equivalent to 0.01g mesalazine is dissolved in 10 ml of absolute ethanol and 30 ml distilled water, after filtration of the solution, the volume is completed to 200 ml of distilled water in a volumetric flask to prepare a solution of 50 ppm mesalazine.

Recommended Procedure and Calibration Graph
To a series of 25ml volumetric flasks aliquots covering the range of 10-400µg (0.4-16 ppm) of mesalazine are transferred, 0.5ml of 1N HCl is then added and the mixtures are shaken.Then 0.5ml of 1% sodium nitrite solution is added and the mixtures are allowed to stand for 3 minutes.
Then 0.3 ml of 3% sulphamic acid solution is added and the mixtures are after that 1.5ml of sodium hydroxide solution (1N) is added, then the volumes are completed to the mark with distilled water.After 10 minutes the absorbance are read at 471nm against blank solution, using 1cm matched cells.(Fig. 1) shows the calibration curve which indicates that Beer's law is obeyed over the concentration range 10-300 µg/25ml final volume, i.e., 0.4-12 ppm and concentration above 300µg/25ml gives negative deviation.The molar absorptivity is 2.9480×10 4 l.mol -1 .cm - .Fig. 1: Calibration graph for mesalazine determination using resorcinol as coupling reagent

RESULTS AND DISCUSSION
For the subsequent experiments, 50µg of mesalazine is taken in 25 ml final volumes and absorbance measurements are performed at 471 nm.

Principle of the method
Mesalazine is reacted with excess nitrite in acidic medium to form the corresponding diazonium salt: The residual nitrite (as nitrite acid) which was undesirable due to its side reaction, such as, nitrosation of coupling agent (Bladyga et al., 1999).Therefore, it should be removed by sulphamic acid which reacts more fast than urea: The colored solution formed by coupling diazotized 5-ASA with resorcinol in alkaline medium.

Study of the Optimum Reaction Conditions
The various parameters effecting and related the colour intensity of the dye have been studied and optimum conditions are selected.

Effect of acids on the diazotization
The effect of the amount of different acid (weak and strong) for the diazotization of 5-ASA, have been investigated.The results are indicated that 0.5ml of 1N HCl produces the highest intensity for the dye, so it has been selected in the subsequent experiments(Table 1).

Effect of nitrite amount and time
The color is reached maximum intensity when using 0.5ml of 1% (w/v) sodium nitrite solution with 3 minutes reaction time, it seems that diazotization of 5-ASA is fast

Effect of sulphamic acid amount and time
The presence of unreacted nitrite is undesirable in diazotization reaction.Therefore, it should be removed by sulphamic acid which fastly reacts with nitrite.The results indicated that 0.3 ml of 3% sulphamic acid solution with 3 minutes standing time are considered to be the most suitable(Table 3), and therefore are selected subsequently.

Effect of resorcinol amount
The effect of resorcinol amount on the color intensity of the dye has been studied.From the results, it can be observed that 4 ml of 0.1% resorcinol is the more suitable amount which gives the highest value of absorbance for the azo-dye formed and the highest value of correlation coefficient (Table 4).

Effect of time on color development
The effect of time on the development and stability period of the coloured dye is investigated under the optimum conditions described above for 5-ASA.From the experimental data, it has been noticed that the azo-dye reached maximum absorbance after 10 minutes and remains stable at least for another 50 minutes when the concentrations of 5-ASA was ≤ 50 µg/25ml.But it was stable for only 30 minutes when the concentration of 5-ASA was ≥100 µg/25ml.However, several measurements can be performed in both cases (Table 5).

Effect of surfactant
The results indicated that addition of different types with different amounts of surfactants give no useful effect.Therefore, it has been recommended to eliminate their use in the subsequent experiments (Table 6).

Effect of base
The preliminary experiments have shown that diazotized 5-ASA gave colored dye of highest intensity with resorcinol in alkaline medium, therefore the coupling reaction has been carried out with different (strong and weak) bases and the results show that sodium carbonate and sodium bicarbonate gave better sensitivity than sodium ydroxide and potassium hydroxide.But the later bases gave better color contrast (∆λ), and the azo-dye formed has good stability compared with weak bases, so that 1.5ml of 1N sodium hydroxide solution has been recommended for the subsequent experiments (Table7).

Final absorption spectra
When mesalazine is treated according to the recommended procedure, the absorption spectrum shows a maximum absorption at 471 nm, characteristic of the orange dye .The reagent blank shows nill absorption at the wavelength of maximum absorption (Fig. 2).

Accuracy and Precision
Three different concentrations of 5-ASA are used in the determination of the accuracy and precision of the method, the results shown in Table 8 indicate that the method has good accuracy and precision.

Nature of the Dye
The composition of the intense orange dye that results from the reaction ofdiazotized 5-ASA with resorcinol has been established using the continuous variations and the mole-ratio methods, the results indicate that the dye has a combination 1:1 ratio of diazotised 5-ASA to recorcinol (Fig. 3 and 4).Orange azo-dye

Interference
The effect of some foreign compounds which often accompanied pharmaceutical preparations were studied by adding four different amounts (50, 200, 500 and 1000µg) to 50µg mesalazine in a final volume 25ml (Table 9).The results in table indicated that the studied foreign compounds do not interfere in the determination of mesalazine using the proposed method.An error not more than of 5.1% in the absorbance readings is considered tolerable from that of the mesalazine alone.

Application of the method
To test the applicability of the present method, it has been applied to the determination of 5-ASA in pharmaceutical preparation (capsules).On applying proposed procedure, good recovery is obtained as shown in Table 10.

Evaluation of the proposed method
Because there is no standard method in the literature for determination mesalasine, so that this standard addition method applied in order to prove that the proposed method can be used in the determination of mesalazine without interferences.(Table 11 and Fig. 5).The results in Table 11 and Fig. 5 indicated that the proposed method can be used to determine mesalazine in pharmaceutical preparation (capsules) with satisfactory results.

Comparison of Methods
Table 12 shows the comparison between the analytical variables obtained from the present method with those of recent spectrophotometric method.The proposed method is simple, rapid, sensitive and do not need any pretreatment of mesalazine or extraction of the dye formed and has good precision.

Fig. 2 :
Fig. 2: Absorption spectra of 50 µg mesalazine / 25 ml treated according to the recommended procedure and measured against (A) reagent blank, (B) distilled water and (C) reagent blank measured against distilled water

Fig. 5 :
Fig.5: Graphs of standard addition method for the determination of mesalazine in pharmaceutical preparations (capsules).

Table 1 :
Effect of acid on absorbance

Table 2 :
The effect of sodium nitrite amount and time on absorbance

Table 3 :
The effect of sulphamic acid amount and time on absorbance

Table 4 :
The effect of resorcinol amount

Table 5 :
The effect of time on absorbance Absorbance

Table 6 :
Effect of surfactant.

Table 8 :
Accuracy and precision of the method

Table 9 :
Effect of foreign compounds for assay of mesalazine Recovery

Table 10 :
Application of the method

Table 11 :
The results of standard addition method