Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 74 No. 2 pp.

401ñ404, 2017 ISSN 0001-6837

Polish Pharmaceutical Society

DEVELOPMENT AND VALIDATION OF BROMATOMETRIC,

DIAZOTIZATION AND VIS-SPECTROPHOTOMETRIC METHODS FOR
THE DETERMINATION OF MESALAZINE IN PHARMACEUTICAL
FORMULATION
ELØBIETA ZAWADA, ELØBIETA PIRIANOWICZñCHABER, ALEKSANDER SOMOGI
and TOMASZ PAWI—SKI*

Department of Drug Chemistry, Medical University of Warsaw, 1 Banacha St., 02-097 Warszawa, Poland

Abstract: Three new methods were developed for the quantitative determination of mesalazine in the form of
the pure substance or in the form of suppositories and tablets - accordingly: bromatometric, diazotization and
visible light spectrophotometry method. Optimizing the time and the temperature of the bromination reaction
(50∞C, 50 min) 4-amino-2,3,5,6-tetrabromophenol was obtained. The results obtained were reproducible, accu-
rate and precise. Developed methods were compared to the pharmacopoeial approach - alkalimetry in an aque-
ous medium. The validation parameters of all methods were comparable. Developed methods for quantification
of mesalazine are a viable alternative to other more expensive approaches.

Keywords: mesalazine, bromatometric assay, spectrophotometry, diazotization, validation

Mesalazine (5-ASA) is chemically known as 5- chemoprevention action of 5-ASA which is based

amino-2-hydroxybenzoic acid. It is a derivative of
salicylic acid and the active metabolite of sul-
fasalazine (1). Mesalazine is administered orally or
rectally in the treatment of ulcerative colitis and
Crohnís disease (2-4). There are also reports about
the possibility of using this drug in the treatment of
colorectal cancer (5-7).
Mesalazine causes an anti-inflammatory effect.
The therapeutic mechanism of mesalazine activity is
achieved via the local treatment of the gastrointesti-
nal tractís mucosa. Numerous, nonexclusive mecha-
nisms of action of that compound were described in
literature (2, 8, 9). The anti-inflammatory effect of
mesalazine is achieved by the process of inhibiting
the conversion of arachidonic acid in the mucosa, by
inhibiting cyclooxygenase enzyme.
Mesalazine also inhibits the formation of per-
oxides in the rectal mucosa and inhibits the enzyme
5-lipoxygenase hindering the synthesis of
leukotrienes in the colon. In addition, mesalazine
has a similar effect to activate PPAR-γ nuclear
receptors located in the intestinal epithelial cells,
reducing the activity of transcription factor NF-κB
that regulates the expression of genes for pro-
inflammatory proteins (10). There are also reports of
on inhibition of protein phosphatase 2A (PP2A).
PP2A is an enzyme involved in the phosphorylation
and degradation of β-catenin leading to inactivation
of Wnt/β-catenin which regulates many important
processes in the organism i.e., carcinogenesis, apop-
tosis or cell proliferation (11).
Currently, several approaches has been
described for determination of mesalazine in the
form of a pharmaceutical formulation. Various
instrumental methods were described such as: spec-
trofluorometric, UV spectrophotometric, differential
pulse voltammetry, adsorptive stripping voltamme-
try, micellar electrokinetic chromatography, high
performance liquid chromatography, ultra-perform-
ance liquid chromatography, liquid chromatography
with tandem mass spectrometry and pharmacopoeial
titrimetric method - alkalimetry in an aqueous medi-
um (12-15).
Most of the above methods, despite the high
level of application, requires access to expensive
equipment and reagents. The procedure of sample
preparation is often labor intensive and requires
qualified staff.
Based on the properties resulting from the
chemical structure of mesalazine new, rapid and

* Corresponding author: e-mail: tomasz.pawinski@wum.edu.pl.

401
402 ELØBIETA ZAWADA et al.
more economical methods for the quantitative deter-
mination of mesalazine in pharmaceuticals were
developed: bromatometric assay, diazotization
method and visible light spectrophotometry.
EXPERIMENTAL
Materials and reagents
All used reagents were of analytical reagent
grade and solutions were made in distilled water.
Pharmaceutical grade mesalazine certified to be
more than 98 % pure (Tokyo Chemical Industry),
and Asamax 500 enteric-coated tablets, and Asamax
250 suppositories (Astellas Pharma) were purchased
from local market.
Apparatus and methods
A Specol spectrophotometer with 1 cm quartz
cells was used for all absorbance measurements.
Mesalazine was converted to a stable, purple-blue
derivative - a complex of ferric chloride (III).
The concentration range of the calibration
curve was 0.01 ñ 0.08 mg/mL. The wavelength was
530 nm.
Bromination reactions were carried out in a
water bath. Potentiometric measurements were per-
formed using a platinum electrode (diazotization
method) and glass electrode (alkalimetry).
General procedures
Bromatometric method
Accurately weighed about 0.03 g of the sub-
stance was dissolved in 50 mL of water at 50OC in a
stoppered conical flask. Then, there were added 10
mL of HCl (281.0 g/L), 0.1 g of KBr and 50.0 mL of
KBrO3 (0.0167 mol/L) RM. The solution was rest-
ing for 50 min in 50OC, in the dark. Then, there were
added 15 mL of a solution of KI (100 g/L). After 15
min, to the solution was added 50 mL of CH3COOH
(856 g /L), and the liberated iodine titrated with a
solution of Na2S2O3 (0.1 mol/L) RM. 2 mL of a solu-
tion of starch was added at the end of the titration.
Control example was processed similarly.
Calculations were based on the relationship that
1 mole of mesalazine reacted with 4 moles of the
atomic bromine and 1 mL of potassium bromate (0.1
mol/L). RM corresponds to 0.001914 g of mesalazine.
Procedure for tablets
Tablets were weighed and pulped into a fine
powder. An accurately weighed amount containing
0.03 g of mesalazine was transferred to a volumetric
flask, dissolved in 50 mL water (50OC) and analyzed
according to the previously described procedure.
Procedure for suppositories
The suppositories were weighed and shredded.
An accurately weighed quantity containing 0.03 g of
mesalazine was transferred to a volumetric flask,
dissolved in 50 mL water (50OC) and analyzed
according to the previously described procedure.
Diazotization method
Accurately weighed about 0.15 grams of the
substance was dissolved in 40 mL of HCl (161 g/L)
and boiled for one minute. After cooling (down to
about 2-5OC) the mixture was slowly titrated with a
solution of NaNO2 (0.1 mol/L) RM. The end point
of the titration was determined potentiometrically
using a platinum electrode.
Calculations were based on the relationship
that 1 mole mesalazine is reacted with 1 mole of
NaNO2. 1 mL of sodium nitrite solution (0.1 mol/L)
RM corresponds to 0.01531 g of mesalazine.
Procedure for tablets
Tablets were weighed and pulped into a fine
powder. An accurately weighed quantity containing
0.15 g of mesalazine was dissolved in 40 mL of HCl
(161 g/L) and boiled for 1 min. After cooling, the
solution was analyzed according to the previously
described procedure.
Procedure for suppositories
The suppositories were weighed and shredded.
An accurately weighed quantity containing 0.15 g of
mesalazine was dissolved in 40 mL of HCl (161
g/L) and boiled for 1 min. After cooling, the solution
was analyzed according to the previously described
procedure.
Visñspectrophotometric method
Calibration curve
Accurately weighed approximately 0.1 g of
mesalazine standard was dissolved in 90 mL of boil-
ing water in a volumetric flask. After cooling down
to the ambient temperature, the flask was filled up
with water to 100 mL. Solutions of the following
concentrations were made: 0.08, 0.06, 0.04, 0.02
and 0.01 mg/mL. To 25 mL of each solution was
added 1 mL of FeCl3 (0.05 g/L), and the absorbance
was measured. The following calibration curve was
obtained: y = 11.578x + 0.0039.
Accurately weighed approximately 0.1 g of
mesalazine was dissolved in 100 mL of boiling
water in a volumetric flask with a capacity of 100.0
mL. After cooling down to the ambient temperature,
5.0 mL of the solution was transferred to a volumet-
ric flask with a capacity of 100.0 mL and filled up to
 Development and validation of bromatometric, diazotization and... 403

mark with water. Then, to 25.0 mL of obtained mix-
ture was added 1 mL of FeCl3 (0.05 g/L), and after
that the absorbance was measured.
Procedure for tablets
Tablets were weighed and pulped into a fine
powder. An accurately weighed amount containing
0.10 g of mesalazine was dissolved in 100 mL of
boiling water. After cooling down, 5.0 mL of the
solution was transferred to a flask with a capacity of
100.0 mL and filled up to mark with water. Then, to
25.0 mL of obtained mixture was added 1 mL of
FeCl3 (0.05 g/L) and the absorbance was measured.
Procedure for suppositories
The suppositories were weighed and shredded.
An accurately weighed amount containing 0.10 g of
mesalazine was dissolved in 100 mL of boiling
water. After cooling, 5.0 mL of the solution was
transferred to a volumetric flask with a capacity of
100.0 mL and filled up to mark with water. Then, to
25.0 mL of obtained mixture was added 1 mL of
FeCl3 (0.05 g/L), and the absorbance was measured.
Alkalimetric method
The quantitative analysis was performed in
accordance with the prescription indicated in FP X
(15). Accurately weighed approximately 0.05 g of
mesalazine was dissolved in 100 mL of boiling
water. After cooling down, solution was titrated
with NaOH solution (0.1 mol/L) RM. Potentiometer
was used to determine the end point of the titration.
Calculations were based on the relationship that 1
mole of mesalazine reacts with one mole of NaOH
and 1 mL of sodium hydroxide solution (0.1 mol/L)
RM corresponds to 0.01531 g of mesalazine.
Procedure for tablets
Tablets were weighed and pulped into a fine
powder. An accurately weighed amount of triturated
tablets, containing 0.05 g of mesalazine, was trans-
ferred to a volumetric flask, dissolved in 100 mL of
boiling water and analyzed according to the previ-
ously described procedure.
RESULTS AND DISCUSSION
Validation
Validation of the proposed methods was per-
formed by determination of ten replicates of fixed
concentration of the drug (in tablets, suppositories
and pure substance). From obtained results were
appointed following values: arithmetic mean, stan-
dard deviation S, relative standard deviation RSD
%, coefficient of variation CV% and deviation from
the values declared by the manufacturer was calcu-
lated. The readings are shown in Table 1.
The developed method gave reproducible
results. Statistical analysis confirmed the usage
validity of each approach in the quantitative deter-
mination of mesalazine. The results of the analysis
proved to be very similar to the results obtained
using the reference method.
The results obtained during studies are within
the acceptable range of tolerance for the average
content of the active ingredient. This demonstrates
accuracy of the described analytical methods.
Diazotization approach is the most accurate -
the results were very similar to the values declared
by the manufacturer. On the contrary, the lowest
accurate was achieved by determining mesalazine in
suppositories. The relatively large deviation in the

Table 1. The validation parameters of developed methods.

Mean RSD CV Accuracy

Method S
[%] [%] [%] [%]
Pure substance 100.12 0.5017 0.0050 0.50 0.12
Bromatometric Tablets 100.67 1.1769 0.0117 1.17 0.67
Suppositories 95.93 0.7328 0.0076 0.76 4.07
Pure substance 100.04 0.1572 0.0016 0.16 0.04
Diazotization Tablets 99.58 1.9946 0.0200 2.00 0.42
Suppositories 99.28 0.3602 0.0036 0.36 0.72
Pure substance 99.45 0.4263 0.0043 0.43 0.55
Spectrophotometric Tablets 99.34 0.6764 0.0068 0.68 0.66
Suppositories 90.04 2.2500 0.0250 2.50 9.96
Pure substance 99.91 0.6530 0.0065 0.65 0.09
Alkalimetric
Tablets 99.97 0.1200 0.0012 0.12 0.03
404 ELØBIETA ZAWADA et al.
spectrophotometric method (9.96%) was possibly a
result of experimenter mistakes or mesalazine
adsorption on the surface of congealed suppository
mass.
In any case, the CV coefficient did not exceed
2.5%, which proves high precision methods.
Bromatometric, diazotization and visible spec-
trophotometric methods are characterized by high
reproducibility of test results of mesalazine in pure
form. CV values are in the range from 0.16% to
0.50% and are less than the reference method,
wherein CV is 0.65%.
Results of the determination of 5-aminosali-
cylic acid in tablets obtained by bromatometric and
alkalimetric methods (CV 1.17% and 2.00%) were
less reproducible than those obtained by visible
spectrophotometric method (CV 0.68%).
The best results of reproducibility for supposi-
tories gives titration methods (CV bromatometric
and diazotization methods: 0.76% and 0.36%). In
the spectrophotometric approach, the coefficient of
variation was high (2.50%). The results obtained by
the alkalimetric method were not reproducible and
not accurate, thus they were not validated.
The relationship between the concentration of
mesalazine in the reference samples and the
absorbance (λ = 530 nm - spectrophotometric
method) is a linear function with a relatively high
coefficient of determination R2 = 0.9905, which indi-
cates a high correlation of results.
CONCLUSION
Validation parameters of developed analytical
methods are comparable with pharmacopeial refer-
ence method. The results indicate that proposed
methods are accurate, precise and can be used for
the routine determination of mesalazine in pure sam-
ples and pharmaceutical formulations. Established
methods do not require expensive or specialized
equipment and regents, therefore, they can be wide-
ly used in many laboratories as an alternative to
other less economical methods.
REFERENCES
1. Azad Khan A., Piris J., Truelove S.: Lancet 2,
8044 (1977).
2. Rainsford K.D.: Aspirin and Related Drugs,
Taylor & Francis, London & New York 2004.
3. Moshkovska T., Mayberry J.F: World J.
Gastroenterol. 13, 32 (2007).
4. Lim W.Ch., Hanauer S.: Cochrane Database
Syst. Rev. 8, 12 (2010).
5. Kim Y.H., Kim M.H., Kim B.J., Kim J.J.,
Chang D.K. et al.: Dig. Liver Dis. 41, 5 (2009).
6 Velayos F.S., Terdiman J.P., Walsh J.M.: Am. J.
Gastroenterol. 100, 6 (2005).
7. Narisawa T., Fukaura Y.: Dis. Colon Rectum
46, 7 (2003).
8. Sza≥ek E.: Farmacja WspÛ≥czesna 8 (2015).
9. Lauritsen K., Laursen L.S., Bukhave K., Rask-
Madsen J.: Gastroenterology 9, 4 (1986).
10. Desreumaux P., Ghosh S.: Aliment. Pharmacol,
Ther. 24 (Suppl. 1), 2 (2006).
11. Lamparska-Przybysz M., Wieczorek M.,
Majorek M., Guzenda P.: WspÛ≥czesna
Onkologia 10, 10 (2006).
12. Altayib F., Abdalla A., Elbashir A.: Med.
Chem. 4, 3 (2014).
13. Moharana A.K., Banarjee M., Panda S., Muduli
J.N.: Int. J. Pharm. Pharm. Sci. 3, 2 (2011).
14. Nandipati S., Reddy V.K., Reddy T.R.: Int. J.
Pharm. Pharm. Sci. 5, 1 (2013).
15. Polish Pharmacopoeia X, Urzπd Rejestracji
ProduktÛw Leczniczych, WyrobÛw Medycz-
nych i ProduktÛw BiobÛjczych, Warszawa
2014.
Received: 29. 08. 2016