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ORIGINAL ARTICLE
Analytical Chemistry Department, Faculty of Pharmacy, Beni-Suef University, Hussin El-Shafey Street, Beni-Suef City, Egypt
Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, Kasr El-Aini Street, 11562, Cairo, Egypt
KEYWORDS
Nitazoxanide;
Degradation;
Derivative
spectrophotometry;
Chemometrics;
TLC-densitometry;
Stability-indicating
methods
Abstract Three sensitive, selective and reproducible stability-indicating methods are presented for
determination of nitazoxanide (NTZ), a new anti-protozoal drug, in presence of its degradation
products. Method A utilizes the rst derivative of ratio spectra spectrophotometry by measurement of
the amplitude at 364.4 nm using one of the degradation products as a divisor. Method B is a
chemometric-assisted spectrophotometry, where principal component regression (PCR) and partial
least squares (PLS) were applied. These two approaches were successfully applied to quantify NTZ in
presence of degradation products using the information included in the absorption spectra in the range
260360 nm. Method C is based on the separation of NTZ from its degradation products followed by
densitometric measurement of the bands at 254 nm. The separation was carried out on silica gel 60F254,
using chloroformmethanolammonia solutionglacial acetic acid (95:5:1:1 by volume, pH 5.80) as a
developing system. These methods are suitable as stability-indicating methods for the determination of
NTZ in presence of its degradation products either in bulk powder or in pharmaceutical formulations.
Statistical analysis of the results has been carried out revealing high accuracy and good precision.
& 2012 Xian Jiaotong University. Production and hosting by Elsevier B.V. All rights reserved.
1.
Introduction
106
Figure 1
2.
2.1.
Experimental
Instruments
A double beam UV/VIS spectrophotometer (Shimadzu,
Japan) model UV-1601 PC with a quartz cell of 1 cm path
length. The spectral band width is 2 nm and the wavelength-scanning speed is 2800 nm/min. All data analysis
was performed using PLS-Toolbox 2.0 running under
MATLABs, version 6.5 [25].
TLC scanner 3 densitometer (Camag, Muttenz, Switzerland).
The following requirements are taken into consideration:
J Slit dimensions: 5 0.2 mm
J Scanning speed: 20 mm/s
J Spraying rate: 10 s/mL
J Data resolution: 100 mm/step
J Band width: 6 mm
J Result output: Chromatogram and integrated peak area
2.2.
107
separated degradation products were subjected to IR and mass
spectral analyses for subsequent identication.
2.3.
Samples
Pharmaceutical formulations
s
2.4.
Degraded sample
Standard solutions
2.2.3.
Procedure
108
Figure 2 IR spectra of nitazoxanide (A), Deg I (tizoxanide) (B), 5-nitro-1,3-thiazol-2-amine (Deg II) (C) and salicylic acid (Deg III) (D).
Chemometric methods
109
110
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
Concentration (mg/mL)
NTZ
Deg I
Deg II
Deg III
6
10
6
4
4
8
10
8
6
10
10
2
8
2
6
8
8
4
2
4
6
2
2
10
4
10
6
4
10
4
8
8
6
3
3
2
2
4
5
4
3
5
5
1
4
1
3
4
4
2
1
2
3
1
1
5
2
5
3
2
5
2
4
4
3
5
3
2
2
4
5
4
3
5
5
1
4
1
3
4
4
2
1
2
3
1
1
5
2
5
3
2
5
2
4
4
3
5
4
3
2
4
5
4
3
5
5
1
4
1
3
4
4
2
1
2
3
1
1
5
2
5
3
2
5
2
4
4
3
5
4
5
111
Deg III
Deg I
Symmetry factor
Resolution (Rs)
Capacity factor (K0 )
Selectivity (a)
1.01
1.21
1.20
2.04
0.98
Deg II
1.05
2.57
2.64
5.12
2.46
NTZ
1.02
4.55
7.35
4.77
3.3.
Chemometric methods
112
Table 3 Determination of nitazoxanide in laboratory prepared mixtures by rst derivative of ratio spectra spectrophotometric
method and TLC-densitometric method.
% Degradation
products
10
25
35
45
55
70
90
Mean7SD
DD1 (mg/mL)
TLC (mg/band)
DD1
TLC
18:1:0.5:0.5
15:2.5:1.3:1.2
6.5:1.5:1:1
11:4.5:2.5:2
9:6:2.6:2.4
6:6.7:3.8:3.5
1.8:0.1:0.05:0.05
1.5:0.3:0.1:0.1
1.3:0.3:0.02:0.02
1.1:0.45:0.25:0.2
0.9:0.5:0.3:0.3
0.6:0.65:0.4:0.35
0.2:0.9:0.5:0.4
100.56
99.00
100.43
101.17
98.00
101.00
100.0371.25
100.44
99.13
100.83
100.89
97.60
101.50
100.26
100.0971.32
Percent of the degradation products were calculated according to their molecular weight.
a
Average of 3 determinations.
Table 4 Results of determination of nitazoxanide, Deg I, Deg II and Deg III in the validation set using the proposed multivariate
calibration methods.
Mixture no.
Recoverya (%)
NTZ
1
2
3
4
5
6
7
8
9
10
11
12
Mean7SD
a
Deg I
Deg II
PCR
PLS
PCR
PLS
PCR
PLS
PCR
PLS
100.14
101.43
99.54
97.87
100.63
98.93
100.53
99.10
98.23
97.79
100.58
101.34
99.6871.29
100.11
101.30
99.65
97.91
100.61
98.95
100.50
99.16
98.25
97.82
100.50
101.26
99.6771.24
101.34
102.25
100.78
101.41
100.69
98.35
99.52
101.06
102.17
100.78
97.75
96.78
100.2471.76
101.33
102.20
100.73
101.40
100.67
98.36
99.52
101.05
102.11
100.77
97.75
96.91
100.2371.72
98.32
96.71
98.38
102.34
100.86
99.46
98.61
103.10
102.77
101.31
97.58
103.23
100.2272.32
98.50
96.83
98.43
101.88
100.82
99.46
98.65
103.04
102.80
101.30
97.75
102.86
100.1972.19
102.43
100.78
99.64
101.63
102.59
103.15
96.80
98.47
99.31
98.32
97.40
102.88
100.2872.25
102.05
100.71
99.75
101.6
102.33
102.88
97.25
98.47
99.35
98.33
97.47
102.75
100.2572.08
Average of 3 determinations.
3.4.
Deg III
TLC-densitometric method
Chromatographic techniques overcome the problem of overlapping absorption spectra of mixture of drugs or in presence
Determination of nitazoxanide in pharmaceutical formulations by the proposed methods and application of standard addition technique.
Pharmaceutical
formulation
DD1 method
Taken
(mg/
mL)
Chemometric methods
Founda
7SD (%)
PCR
Nit cleans
tablets
Nitazods oral
suspension
8.00
8.00
100.78
70.96
98.58
71.07
98.76
71.01
6.00
99.00
8.00
10.00
12.00
101.25
101.00
101.00
6.00
101.33
8.00
10.00
12.00
98.63
99.10
101.25
6.00
101.67
8.00
10.00
12.00
100.63
100.70
99.25
100.50 4.00
71.02
100.08 4.00
71.41
100.56 4.00
70.99
PLS
TLC-densitometric method
Pure
Recoveryb (%)
added (mg/
mL)
PCR Mean PLS
7SD
Taken
(mg/
mL)
Mean
7SD
99.90
71.19
99.33
Founda Pure
Recoveryb Mean
7SD
added (mg/ (%)
7SD
(%)
mL)
99.93 0.800
71.00
100.75
101.00
98.50
101.05
70.89
98.94
70.91
100.75
100.80
98.83
97.58
71.05
0.600
100.33
0.800
1.000
1.200
100.88
101.00
99.25
0.600
101.67
0.800
1.000
1.200
98.63
99.30
99.25
0.600
101.33
0.800
1.000
1.200
98.88
101.10
100.83
100.37
70.80
Table 5
99.72
71.34
100.54
71.12
Average of 6 determinations.
Average of 3 determinations.
113
114
Table 6 Assay parameters and method validation obtained by applying the proposed methods for the determination of
Nitazoxanide.
Parameters
DD1
spectrophotometric
method
Chemometric methods
PCR
TLC-densitometric
method
PLS
Range
224 mg/mL
0.42.0 mg/band
Linearity
Slope
Intercept
Correlation coefcient (r)
Accuracy (Mean7SD)
Specicity and selectivity
0.0480
0.0010
0.9999
99.8570.75
99.8871.21
0.9972
0.0017
0.9996
99.6871.29
0.9973
0.0108
0.9997
99.6771.24
2.2575
0.1262
0.9998
100.0771.15
100.0771.44
Precision (%RSD)
Repeatabilitya
Intermediate precisionb
LODc
0.83
0.96
0.95 mg/mL
0.75
0.77
0.78 mg/mL
0.68
0.84
0.83 mg/mL
0.81
1.12
0.10 mg/band
210 mg/mL
The intraday (n 3), average of three different concentrations repeated three times within day.
The interday (n 3), average of three different concentrations repeated three times in three successive days.
c
Limit of detection is determined experimentally for TLC-densitometric method and via calculations for the other methods.
b
r 0:9998
Stability-indication
115
Table 7 Statistical comparison of the results obtained by the proposed methods and the reported method for the determination
of pure nitazoxanide.
Parameters
Mean
SD
% RSD
n
Variance
Students t-test
F-value
DD1 spectrophotometric
method
99.85
0.75
0.75
6
0.56
0.09 (2.18)
2.48 (4.88)
Chemometric methods
PCR
PLS
99.68
1.29
1.29
12
1.66
0.31 (2.10)
1.19 (3.60)
99.67
1.24
1.24
12
1.54
0.34 (2.10)
1.11 (3.60)
TLC-densitometric
method
Reported
methoda [23]
100.07
1.15
1.15
6
1.32
0.23 (2.18)
1.05 (4.88)
99.90
1.18
1.18
8
1.39
Figures between parenthesis represent the corresponding tabulated values of t and F at P 0.05.
a
RP-HPLC using 0.1% phosphoric acidacetonitrile (44:45, v/v) at pH 6 with UV detection at 240 nm and ow rate 1 mL/min.
4.
Conclusion
Method validation was performed according to ICH [24] guidelines for the proposed methods. Table 6 shows the results of
accuracy, repeatability and intermediate precision of the methods.
Other regression equation parameters are shown in Table 6, which
shows good linear relationship for the methods as revealed by the
correlation coefcient. Results of assay validation parameters of
the proposed chemometric methods are demonstrated in Table 6,
where satisfactory correlation coefcient (r) value was obtained for
NTZ in the validation set by PCR and PLS optimized models
indicating good predictive abilities of the models.
3.8.
References
Method validation
Statistical analysis
116
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