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 (R2 = 0.999) with limit of detection (LOD) being 0.113 µg/ml. The molar absorptivities were  1.5.104 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

Dung, Nguyen Hoang

Dung, Nguyen Trung

Lan Anh, Nguyen Thi

Oanh, Dong Thi

Vietnam Academy of Science and Technology: Journals Online

 
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, diphenylamine…to 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 
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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.
 
 


Development of spectrophotometric method for determination of mesalazine in pharmaceutical preparation

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Development of spectrophotometric method for determination of mesalazine in pharmaceutical preparation

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