Bangladesh Pharmaceutical Journal 15(1): 47-51, 2012 Development and Validation of a Simple and Rapid UV Spectrophotometric Method for Assay of Nitazoxanide in Pharmaceutical Dosage Forms Fahima Aktar1, Md. Ruhul Kuddus1, Akter Hossen2, Md. Khalid Hossain1 and Mohammad A. Rashid1
1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Dhaka, Dhaka-1000, Bangladesh
2Department of Pharmacy, State University of Bangladesh, 77, Satmosjid Road, Dhanmondi, Dhaka-1207, Bangladesh
Abstract This paper reports the development and validation of a simple UV spectrophotometric method for the assay of nitazoxanide in tablets and powder for oral suspension. The method was linear in the range of 5 and 50 µg/ml presenting a good correlation coefficient (r = 0.9996, n = 7). Precision and accuracy analyses showed low relative standard deviation (< 2.50%) and good recoveries (94.01-113.9%). The procedure was found to be accurate, precise, linear, robust, simple and cost effective. It did not require any polluting reagents and can be applied to assay of nitazoxanide in pharmaceutical dosage forms notably tablets and powder for oral suspensions. Key words: Nitazoxanide, tablets, suspensions, UV spectrophotometry. Introduction
Nitazoxanide (NTZ) is a new antiparasitic and
antiprotozoal agent having broad-spectrum of activity. It is
a nitrothiazole derivative and its chemical name is
2-acetyloxyl-N-(5-nitro-2-thiazolyl) benzamide (Fig. 1)
(Rossignol et al., 1976). NTZ was first described in 1975
by Jean Francois Rossignol and was initially developed as
a veterinary anthelminthic with activity against intestinal
Although no official method for the determination of
nematodes, cestodes and trematodes. NTZ was approved
this drug in oral formulation has been described yet, NTZ
by the US Food and Drug Administration (FDA) in 2002
in pharmaceutical formulations (tablets and powder for
for use in human beings (Fox et al., 2005). It is used for
oral suspension) can be estimated by liquid
treating both intestinal protozoal infections and chromatography (LC) (Gopu et al., 2007; Jadhav et al.,
helminthiasis (Raether et al., 2003). It is also used for
2007; Rane et al., 2008; Malesuik et al., 2008), LC-mass
treating diarrhea caused by Giardia lamblia as well as for
spectrometry (LC-MS) (Stockis et al., 1996; Zhao et al.,
cryptosporidiosis in immune-compromised patients, 2008), high performance-thin layer chromatography (HP-
including those with AIDS or HIV infection (Cravier et
TLC), RP-HPLC and UPLC (Kalta et al., 2008). Although
al., 1978; O'Neil et al., 2001; Murphy et al., 1985; Stockis
various analytical methods have been reported for
et al., 1996; Rossignol et al., 1984).
determination of NTZ in bulk as well as in pharmaceutical
NTZ is a light yellow/pink crystalline powder that is
formulations, the reported chromatographic methods
poorly soluble in ethanol, practically insoluble in water
necessitate sample pretreatment and time-consuming
with a molecular mass of 307.283 gm/mole and molecular
extraction steps prior to analysis of the drug (Sakamoto
et al., 2008). Several of these methods require the use of
12H9N3O5S. After ingestion, it is converted to
the active metabolites tizoxanide and tizoxanide hazardous and expensive chemicals, which make the glucuronide. In plasma, more than 99% of NTZ is bound
process not only a challenge for the environment but also
to proteins. It is available in the market as tablets and oral
complex. Moreover, these methods require expensive
equipments and considerably skilled personnel.
Correspondence to: Mohammad A. Rashid; Tel.: 880-2-9661900-73, Extn.- 8130, 8131, 8137; Fax: 880-2-8615583; E-mail: [email protected]Aktar et al. /Bangladesh Pharmaceutical Journal 15(1): 47-51, 2012
In this paper, we describe a simple UV- specto-
dispersion of the drug. Then the flasks were shaken
photometric method for the determination of NTZ in tablet
ultrasonically for 20.0 minutes and the solution was
and powder for suspension formulations. The method has
filtered through Whatman no.1 filter paper (Whatmann
been optimized and validated as per the ICH guidelines
International Limited, Kent, UK). The residue was washed
well with ethanol for complete recovery of the drug. After
filtration an aliquot of 1.0 ml of this solution was
Materials and Methods
transferred into a 50.0 ml volumetric flask and the volume was adjusted upto the mark with ethanol. In case of
Apparatus:A Shimadzu UV-Visible spectrophoto-
powder for oral suspension, at first suspension was made
meter (UVmini-1700, Shimadzu Corporation, Kyoto, by adding purified water (according to direction in the
Japan) with matching quartz cells was used for all label). From this suspension, 5.0 ml aliquot was taken in
100.0 ml volumetric flask and about 70.0 ml of ethanol
reagents:All chemicals and reagents
was added, sonicated for 20 minutes and diluted up to the
were of analytical or pharmaceutical grade. NTZ was
mark (100.0 ml) with ethanol to get a concentration of 1.0
kindly supplied by Incepta Pharmaceuticals Ltd. mg/ml. The solution was filtered through Whatman no.1 (Bangladesh) and was used as the reference standard. A
filter paper and 1.0 ml of 1.0 mg/ml NTZ was further
standard solution of NTZ was prepared by dissolving 50
diluted to 50 ml in a volumetric flask with ethanol to
mg of NTZ in 50 ml of ethanol, transferred to a 100 ml
produce a final concentration of 20 µg/ml. The assay was
volumetric flask and the volume was adjusted to the mark
completed following the same proposed method used for
with ethanol to obtain a stock solution of 500 µg/ml. Five
different tablets and five powder samples for suspension
of NTZ coded as NTZ-ta to NTZ-te and NTZ-pa to NTZ-
The developed analytical method has been validated
for specificity, linearity, limit of detection, precision,
λmax: An ultra violet spectrophoto-
accuracy and robustness as mentioned below.
metric scanning (190-380 nm) was carried out with the
Specificity: The reference standard and quality control
reference solution (5.0µg/ml) to select the λmax for samples of NTZ were subjected to stress conditions (i.e. detection of nitazoxanide.
light, heat etc). Each stressed sample was measured to
procedure: Aliquots of 500 µg/ml
determine the content of NTZ and the results were
from the standard stock solution of NTZ were pipetted
compared with those for an unstressed time zero reference
into a six series of 50.0 ml volumetric flasks. Then the
solution. The reference assay value for each unstressed
mixture was serially diluted with ethanol to get NTZ
product was evaluated and the contents of degradation in
solutions of 5.0, 10.0, 15.0, 20.0, 25.0 and 50.0 µg/ml.
the stressed and control samples were estimated relative to
The contents of each flask were mixed well and the assay value. The system response was examined for immediately transferred to the spectrophotometric cell.
the interference or overlaps if any, with NTZ responses at
Procedure for the determination of NTZ in Linearity: The linearity was evaluated with six pharmaceutical formulations:The test sample solutions
standard solutions: 5.0, 10.0, 15.0, 20.0, 25.0 and 50.0
containing NTZ at a concentration of 20.0 µg/ml were
µg/ml. The determination was repeated five times at each
prepared. For this 20 tablets from each sample were
concentration level. The linearity was evaluated by linear
weighed and the average weight was calculated. The
regression analysis, which was calculated by the least
contents of 20 tablets were obtained by gently peeling of
the hard shells and then crushed to a fine powder and an
Limits of detection (LOD) and limit of quantification
amount equivalent to 100 mg of NTZ was transferred to
(LOQ):Limits of detection (LOD) and quantitation (LOQ)
each of the 100.0 ml volumetric flask and 70.0 ml of
for the assay were calculated using the following
ethanol was added and left for 10.0 minutes for complete
equations (Mustafa et al., 2000).
Aktar et al. /Bangladesh Pharmaceutical Journal 15(1): 47-51, 2012
LOD = 3.3 ×So /b and LOQ = 10 × So/b, where So and
The intra-day and inter-day precision and accuracy
b are the standard deviation and the slope of the were determined and listed in Tables 3 and 4 respectively. calibration line.
Since all the values of accuracy and % CV are well within
the acceptable range of 15%, the results indicated that the
accuracy of the proposed method were determined by
method is reliable, reproducible and accurate.
replicate analysis (n= 4) of calibration standards at three
Calibration curve of NTZ
concentration levels (5.0, 20.0 and 50.0 µg/ml). Inter-day
precision and accuracy were determined by assaying the calibration standard at the same concentration levels for
four consecutive days. Precision and accuracy were based
on the calculated relative standard deviation (RSD, %) and
coefficient of variance (CV,%) of the observed
concentration as compared to the theoretical one,
respectively. For the method to be precise the %CV
determined at each concentration level, should not exceed
Conce ntration (microgram/ml) Robustness:Robustness of the proposed method was
determined by changing the pH of the medium by ± 0.2
Table 1. Absorbance of NTZ solutions of varying concentrations at
units and by maintaining the solutions at room
temperature (25 ± 2 ºC) for 3 h to test the stability of NTZ
in the working diluent (ethanol at pH 4.5).
test:The potency of the tested tablet and
powder for suspension formulations were determined by
Table 2. Regression data for calibration of NTZ Results and Discussion
A simple and rapid UV spectrophotometric assay
method has been developed and validated for analysis of
nitazoxanide (NTZ) in tablets and powder for suspension.
In order to verify the absence of interference of
Table 3. Intra-day precision and accuracy study of standard NTZ
excipients on the analysis of NTZ tablets and powder for
Intra-day precision and accuracy (n = 4 replicates)
suspension, a sample was prepared with all the excipients
present in the tablets but without the drug (placebo).
Absorption spectra did not show any potential interference
A good linear relationship was evident between the
absorbance and concentration in the range of 5.0 to 50.0
Table 4. Inter-day precision and accuracy study of NTZ
µg/ml (Fig. 2). The correlation coefficient was 0.9996
Intra-day precision and accuracy (n = 4 consecutive days)
(Table 2) indicating good linearity. The representative
Calculated concentration (µg/ml) Mean ± SD
linear equation was Y=0.0469x + 0.0235, calculated by
the least squares method. The limit of quantification
(LOQ) was found as 0.907 µg/ml while the limit of
Aktar et al. /Bangladesh Pharmaceutical Journal 15(1): 47-51, 2012 Table 5. Extinction coefficient of NTZ Conclusion
The results obtained and the statistical parameters for
determination of NTZ in pharmaceutical dosage forms
demonstrated that the proposed UV spectrophotometry
method is simple, accurate, fast and precise. The method
showed high sensitivity, acceptable linearity and accuracy.
Therefore it could be easily used for the analysis of pure
drug. Moreover, the method uses simple reagents with
In addition, the reliability of the proposed method was
minimum steps and time for sample preparation, which
also evaluated by means of the determination of the
allow it to be useful for routine analyses and quality-
extinction coefficient of NTZ using Beer-Lambert’s Law
control assays of NTZ in tablets and powder for
After the validation of the newly developed UV
spectrophotometric method, the potency of marketed References formulations was determined by the proposed validated
method and the results are shown in Table 6. Among the
bioanalytical method validation for small molecules. AAPS
different marketed brands used, the potency of all the
Journal. 9, 109-114.
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Table 6. Potency determination of the NTZ formulations
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