FARMACIA, 2008, Vol.LVI, 6
639
SPECTROPHOTOMETRIC DETERMINATION
OF NIMESULIDE THROUGH ION-PAIR
COMPLEX FORMATION WITH
HEXADECYLTRIMETHYLAMMONIUM
BROMIDE
MARINELA FLOREA*, CRINA-MARIA MONCIU, ANDRIŢOIU
MAGDA LAURA, BĂCANU LAVINIA GABRIELA
University of Medicine and Pharmacy “Carol Davila” Bucharest,
Faculty of Pharmacy, Department of Analytical Chemistry, 6th Traian
Vuia str, 020956
*corresponding author: florea.marinela@gmail.com
2
Abstract
The present work reports a new spectrophotometric method for nimesulide assay.
This is based on nimesulide ability to form with hexadecyltrimethylammonium bromide
(cetrimide), in basic media, an ionic association extractible in chloroform, with a maximum
at 404 nm. The method was validated and applied with good results on nimesulide assay in
pharmaceutical dosage forms.
Rezumat
În lucrarea de faţă se prezintă o nouă metodă spectrofotometrică de dozare a
nimesulidului care are la bază proprietatea acestuia de a forma cu bromura de
hexadeciltrimetilamoniu (cetrimid), în mediu bazic, o asociere ionică extractibilă în
cloroform, cu maxim de absorbţie la 404 nm. Metoda a fost validată şi aplicată cu bune
rezultate la determinarea nimesulidului din forme farmaceutice dozate.



ion-pairs
nimesulide
cetrimide
INTRODUCTION
Ion pair formation, initially investigated by the physical chemistry
was found extremely interesting for the chemical analysis, including
pharmaceutical analysis. Modern analytical methods (X-ray spectrometry,
infrared spectrometry, UV-Vis spectrometry, resonance Rayleigh
spectrometry) proved that the formation of ion pairs is a consequence of the
electrostatic, hydrophobic and charge transfer interactions and allowed
optimal experimental conditions setting for their formation [1].
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Nimesulide, 4-nitro-2-phenoxy-methanesulfonanilide (fig. 1) is a
non-steroidal anti-inflammatory drug with antipyretic and analgesic
properties [2].
NHSO2CH3
O
NO2
Figure 1
Chemical structure of nimesulide
The published methods reporting the assay of nimesulide in
pharmaceutical formulations are based on various techniques: fluorimetry
[3], spectrophotometry [4-8], HPLC [9-11], thin layer chromatography [12],
voltametry [13] and capillary elecrophoresis [14].
The present paper describes an extraction spectrophotometric
method for the determination of nimesulide (NS) through ion-pair formation
with cetrimide (CT). The proposed method was validated and applied to the
determination of nimesulide in bulk and in pharmaceutical formulations.
MATERIALS AND METHODS
Instruments
All absorption spectra were registered using a UV-Vis Kary 100
Bio spectrophotometer (Varian Inc.) with a scanning speed of 600 nm/min,
equipped with 10 mm matched quartz cells.
Materials and reagents
All chemicals and reagents were of analytical grade and water has
always been bidistille.
 Nimesulide reference standard (Sigma);
 Nimesulide pharmaceutical substance (Magistra SA);
 Hexadecyltrimetilammonium bromide (Acros Organics);
 Nimesulide stock solution: 0.3559 g reference standard was
dissolved in 10 mL NaOH 10-1M and completed to 100 mL
with water in a volumetric flask;
 Nimesulide work solution: 4 mL stock solution are diluted
with NaOH 10-2 M to 100 mL in a volumetric flask;
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

Cetrimide 6·10-4M prepared by dissolving 0.2180 g
substance in 1000 mL water in a volumetric flask;
NaOH 10-1M and 10-2M solutions prepared by diluting
appropriate volumes of 1M solution
Methods
Procedure for calibration curve
Into a series of 250 mL separating funnels there were measured
accurate aliquots (1–3.5 mL) of the nimesulide work solution, 4 mL
cetrimide solution and NaOH 10-2M to 15 mL. The mixture was extracted
two times, first with 10 mL and second with 5 mL chloroform by shaking
for 1.0 minute, then allowed to stand for clear separation of the two phases.
The chloroform layer was passed through anhydrous sodium sulphate. The
collected extract was transferred and brought to 25 mL with chloroform in a
volumetric flask. The absorbance of the yellow nimesulide – cetrimide (NSCT) ionic association was measured at 404 nm, against corresponding
reagent blank prepared in the same manner, except the addition of the
examined drug (Fig. 2). The calibration graph is linear over the
concentration range 5,69 – 19,93 μg/mL (Fig. 3).
1.2
1
Absorbance
0.8
0.6
0.4
y = 0.0547x - 0.083
0.2
0
0
5
10
15
20
25
Concentration (g/m L)
Figure 2
Absorbance for NS-CT ion-pair
Figure 3
Calibration curve for NS-CT method
Procedure for the assay of dosage forms
Tablets
The contents of 20 tablets (Aulin - 100 mg nimesulide – CSC
Pharmaceuticals) were weighed, ground into a fine powder and mixed. An
accurately weighed portion of the powder corresponding to approximately
45 mg nimesulide (0.1801 g) was transferred into a 250 mL volumetric
flask. 25 mL of NaOH 10-1M were added and after 20 minutes of
mechanically shaking, the suspension was completed to the mark with
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water. After separation (Whatman No. 42 filter paper) appropriate amounts
of filtrate were added in 250 mL separating funnels and the same procedure
was applied as described for the calibration curve.
Granules
The content of 10 sachets of granules for oral suspension (Aulin 100 mg nimesulide – CSC Pharmaceuticals) was weighed, ground into a
fine powder and mixed. An accurately weighed portion of the powder
corresponding to 8 mg nimesulide (0.1597 g) was transferred into a 100 mL
volumetric flask. 10 mL of NaOH 10-1M were added and after 20 minutes of
mechanically shaking, the suspension was completed to the mark with
water. After separation (Whatman No. 42 filter paper) appropriate amounts
of filtrate were added in 250 mL separating funnels and the same procedure
was applied as described for the calibration curve.
RESULTS AND DISCUSSION
The optimization of the method was carefully studied in order to
achieve complete reaction formation, highest sensitivity and maximum
absorbance. In the proposed method, some variables in the reaction conditions
were studied and the influence of these variables on the reaction was tested.
The selection of the optimum wavelength
Absorption spectra of pure nimesulide and cetrimide in NaOH 10-2 M
solutions were obtained at 200 – 800 nm, against water (Figure 4).
The absorption spectra of the ion-pair complex, formed between
nimesulide and cetrimide was measured at 220-800 nm against the blank
solution (Figure 4). The yellow chloroformic extract showed maximum
absorbance at 404 nm. The measurements were made using this wavelength.
Figure 4
Absorbtion spectra of (1) nimesulide (12.11 µg/mL), (2) NS-CT ionic association
(12.11 µg/mL) and (3) cetrimide (12 µg/mL)
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Effect of reagent concentration
The effect of the reagent concentration was studied by measuring
the absorbances of the solutions containing the same concentration of
nimesulide (12 µg/mL) and varied amounts of the cetrimide 6·10-4M
solution. Maximum color intensity of the complex was achieved with 4.0
mL of cetrimide solution. Larger volumes of the reagent had no pronounced
effect on the ion-pair complex formation. The volume of 4.0 mL of
cetrimide 6·10-4 M solution was used for the further determinations.
Stoichiometric relationship
Slope ratio method [15] was used for the stoichiometric
relationship determination. Two series of six extractions of NS-CT ionic
association where made. The first consisted in the extraction of various
amounts of nimesulide (4.40 – 20.16 μg/mL) in the presence of a large and
constant excess of cetrimide (28.84 μg/mL) and the second consisted in the
extraction of various amounts of cetrimide (4.37 – 24.62 μg/mL) in the
presence of a large and constant excess of nimesulide (34.88 μg/mL). The
absorbances of NS-CT ionic association were measured at 404 nm. The
slope of each series of determination was calculated using regression
method (slope1 = 0.04878 and slope2 = 0.05200).
The ratio of the nimesulide and cetrimide in the ion pair was
determined by taking the ratio of the two slopes. The results indicated that
1:1, nimesulide : cetrimide ion-pairs were formed.
Method validation
Accuracy
In order to determine the accuracy of the proposed method, three
levels of drug concentration (10, 12, 15 μg/mL) were analyzed in triplicate
(n=9). Each concentration was obtained from independent stock solutions
prepared using nimesulide pharmaceutical substance. Accuracy was
reported as percent recovery together with the confidence interval (Table 1).
Precision
Precision was determined by studying the repeatability and
intermediate precision. Both were determined using three levels of
nimesulide concentration (the same concentrations used on the accuracy
determination) assessed in triplicate (n=9). Each concentration was obtained
from independent stock solutions prepared using nimesulide pharmaceutical
substance. RSD% values obtained in both cases indicate a good precision
for the proposed method (Table I).
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Limit of detection (LOD) and limit of quantitation (LOQ)
The LOD and LOQ of nimesulide by the proposed method were
determined based on the standard deviation of the blank. An appropriate
number of blank samples (n=6) where analyzed and the standard deviation
of the responses was calculated. LOD and LOQ were calculated as 3.3·σ/S
and 10·σ/S, respectively, where S is the slope of the calibration curve and σ
is the standard deviation of the blank [16].
Table I
Summary of validation data obtained for nimesulide assay
Validation criterion
Parameter
Results
Precision
Repeatability
µ = 99.73 ± 0.5270
RSD% = 0.6873
Intermediate precision
µ = 100.16 ± 0.394
RSD% = 0.5118
Accuracy
µ = 99.78 ± 0.641
Linearity
Regression parameters
Y = 0.0547x – 0.083
Correlation coefficient
0.9999
Range
5.69 – 19.93 μg/mL
Limit of detection (LOD)
0.0452 µg/mL
Limit of quantification (LOQ)
0.137 µg/mL
Analysis of pharmaceutical preparations
The proposed method proved to be selective against excipients
used in the chosen dosage form and has been successfully applied to the
determination of nimesulide in commercial tablets and granules. The results
obtained are summarized in Table II.
Table II
Results obtained in the assay of nimesulide from pharmaceutical preparations
Dosage
Label content
Results*
RSD%
Confidence
form
interval
mg/tb
%
Aulin®
100 mg
99.67
99.67
0.664
99.67 ± 0.5089
tablets
Aulin®
100 mg
99.89
99.89%
0.7358
99.89 ± 0.565
granules
*average of 9 measurements
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CONCLUSIONS
A significant advantage of the extractive spectrophotometric
method consists in the fact that can be applied for the assay of individual
substances in a complex matrix.
The proposed method for the assay of nimesulide, based on the
drug ability to form with cetrimide, in basic media, an ionic association
extractible in chloroform, with a maximum at 404 nm, is simple and not
very expensive. The reagents used in the proposed method are cheaper,
readily available and the procedures do not involve any critical reaction
conditions or tedious sample preparation.
The developed method may be used for routine and quality control
analysis of the investigated drug in dosage forms.
Acknowledgements
This research was supported by a CNCSIS, PNII- Human
Resources –TD project (No. 18/01.10.2007).
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