A comparative study between three stability indicating spectrophotometric methods for the determination of diatrizoate sodium in presence of its cytotoxic degradation product based on two-wavelength selection

Article abstract of DOI:10.1016/j.saa.2015.03.030

Abstract Three sensitive, selective, and precise stability indicating spectrophotometric methods for the determination of the X-ray contrast agent, diatrizoate sodium (DTA) in the presence of its acidic degradation product (highly cytotoxic 3,5-diamino metabolite) and in pharmaceutical formulation, were developed and validated. The first method is ratio difference, the second one is the bivariate method, and the third one is the dual wavelength method. The calibration curves for the three proposed methods are linear over a concentration range of 2-24 μg/mL. The selectivity of the proposed methods was tested using laboratory prepared mixtures. The proposed methods have been successfully applied to the analysis of DTA in pharmaceutical dosage forms without interference from other dosage form additives. The results were statistically compared with the official US pharmacopeial method. No significant difference for either accuracy or precision was observed.

Full text of DOI:10.1016/j.saa.2015.03.030

Accepted Manuscript
A comparative study between three stability indicating spectrophotometric
methods for the determination of diatrizoate sodium in presence of its cytotoxic
degradation product based on two-wavelength selection
Safaa M. Riad, Mohamed K. Abd El-Rahman, Esraa M. Fawaz, Mostafa A.
Shehata
PII:
S1386-1425(15)00326-1
http://dx.doi.org/10.1016/j.saa.2015.03.030
SAA 13444
DOI:
Reference:
Spectrochimica Acta Part A: Molecular and Biomo-
lecular Spectroscopy
To appear in:
Received Date:
Revised Date:
Accepted Date:
3 December 2014
14 February 2015
1 March 2015
Please cite this article as: S.M. Riad, M.K. Abd El-Rahman, E.M. Fawaz, M.A. Shehata, A comparative study
between three stability indicating spectrophotometric methods for the determination of diatrizoate sodium in
presence of its cytotoxic degradation product based on two-wavelength selection, Spectrochimica Acta Part A:
Molecular and Biomolecular Spectroscopy (2015), doi: http://dx.doi.org/10.1016/j.saa.2015.03.030
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A comparative study between three stability indicating spectrophotometric methods
for the determination of diatrizoate sodium in presence of its cytotoxic degradation
product based on two-wavelength selection
Safaa M. Riada,b , Mohamed K. Abd El-Rahmana, Esraa M. Fawaz, Mostafa A.
Shehataa
a
Analytical Chemistry Department, Faculty of Pharmacy, Cairo University,
Kasr-El Aini Street, 11562 Cairo, Egypt.
b
Analytical Chemistry Department, Faculty of Pharmacy, October for Modern
Sciences and Arts University, 6th of October city, Egypt.
* Corresponding author: Esraa M. Fawaz, Analytical Chemistry Department,
Faculty of Pharmacy, Cairo University, Kasr-El Aini Street, 11562 Cairo, Egypt.
E-mail: esraafawaz_2007@yahoo.com. Phone: + 201005639834
Abstract
Three
sensitive,
selective,
and
precise
stability
indicating
spectrophotometric methods for the determination of the X-ray contrast agent,
diatrizoate sodium (DTA) in the presence of its acidic degradation product
(highly cytotoxic 3,5-diamino metabolite) and in pharmaceutical formulation,
were developed and validated. The first method is ratio difference, the second
one is the bivariate method, and the third one is the dual wavelength method.
The calibration curves for the three proposed methods are linear over a
concentration range of 2–24 µg/mL. The selectivity of the proposed methods
was tested using laboratory prepared mixtures. The proposed methods have been
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successfully applied to the analysis of DTA in pharmaceutical dosage forms
without interference from other dosage form additives. The results were
statistically compared with the official US pharmacopeial method.
significant difference for either accuracy or precision was observed.
No
Key words: Sodium diatrizoate, ratio difference, bivariate, dual wavelength, and
stability indicating methods.
1-Introduction
In modern analytical laboratory, there is always a need for significant
stability indicating methods of analysis [1,2]. An ideal stability indicating
method quantifies a drug and resolves its degradation products [3]. The
spectrophotometric methods have the advantages of being the most simple, fast
and applicable in all laboratories, as most of the active compounds show
absorbance in the UV region. But, usually compounds susceptible to degradation
may be present with their degradation products with which they exhibit strongly
overlapping spectra that impede the determination of the main compound.
Diatrizoate sodium (DTA) is one of the most widely used ionic contrast
agents in many medical imaging purposes as it can enhance the image obtained
thanks to its iodine content that can absorb X-rays [4-7]. Reviewing the
literature in hand revealed that there are only two spectrophotometric methods
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and a TLC-spectrodensitometric method for the determination of DTA in
presence of its free amino degradate in spite of its high toxicity [8]. There are
only one direct UV spectrophotometric method [9] and another H1NMR method
for the determination of DTA as a single component [10]. DTA was also
determined in presence of its diiodo degradates by capillary electrophoresis
technique [11] and liquid chromatography [12,13]. DTA is an official drug
whose US pharmacopeial assay method is a precipitimetric titration that depends
on the iodide content in the drug [14].
The goal of this contribution is the development, validation, and
comparison between three different stability indicating spectrophotometric
methods for the determination of DTA in presence of its cytotoxic degradation
product. The three proposed methods, namely, ratio difference, bivariate, and
dual wavelength, are based on careful selection of a pair of wavelengths. They
are applicable to the quality control and routine analysis of DTA in
pharmaceutical preparations.
2-Experimental
2.1. Apparatus
Spectrophotometer:
SHIMADZU
dual
beam
UV-visible
spectrophotometer (Kyoto, Japan), model UV-1650 PC connected to IBM
compatible and a HP1020 laserjet printer. The bundled software, UV-Probe
personal spectroscopy software version 2.21 (SHIMADZU), is used. The
spectral band is 2 nm and scanning speed is 2800 nm/min with 0.1 nm interval.
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2.2. Reference Samples
Diatrizoate sodium reference standard was purchased from Sigma-
Aldrich Co., and its purity was certified to be ≥ 99.99%.
2.3. Pharmaceutical Formulation
Gastrografin® solution manufactured by Schering Company (Belimed,
Spain) batch number 51424A is labeled to contain 0.6 g/mL of diatrizoate
anhydrous base.
2.4. Degraded Samples
10 mL of 2M HCl solution was added to pure DTA (500 mg) in a 250
mL glass-stoppered conical flask and refluxed for
6
hours. Complete
degradation was tested by TLC using chloroform: methanol: ammonium
hydroxide (20: 10: 2 by volume) as a mobile phase. Only one spot was observed
not corresponding to DTA. Subsequently, 2M NaOH solution was added to the
degraded solution till pH was adjusted to about 7, the solution was evaporated
on small flame, and then the degradate is dissolved in methanol, filtered, and left
to evaporate at room temperature. The structure of the isolated degradation
product was elucidated using mass and IR spectrometry.
2.5. Materials and Reagents
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All chemicals used throughout this work were of analytical grade, and distilled
deionized water was used as a solvent (Adwic, Cairo, Egypt).
2.6. Standard Solutions
DTA stock standard solutions with a concentration of 0.1 mg/mL in
distilled water was prepared in 100 mL volumetric flasks by dissolving 10 mg of
pure DTA in distilled deionized water. Degradation product stock solution with
a concentration of 0.1 mg/mL was prepared in distilled deionized water.
2.7. Procedures
2.7.1. Construction of Calibration Graphs
Aliquots (0.2–2.4 mL) of DTA stock solution (0.1 mg/mL) were
transferred into a series of 10 mL volumetric flasks, and the volume was
completed with distilled deionized water. The zero-order spectra were recorded
using distilled deionized water as a blank.
2.7.1.1. Ratio Difference Method
The scanned spectra of the prepared solutions were divided by the
spectrum of 14 µg/mL degradation product. The obtained ratio spectra were
recorded. Calibration curve for DTA was constructed by plotting the difference
between the peak amplitudes of the obtained ratio spectra at 243 and 253 nm
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versus the corresponding concentrations in µg/mL, and the regression equation
was computed.
2.7.1.2. Bivariate Method
The absorbance of pure DTA was measured at 238 and 264 nm and
plotted against the corresponding concentrations, and then the regression
equations were computed at the selected wavelengths.
2.7.1.3. Dual Wavelength Method
Calibration curve for DTA was constructed by plotting the difference
between absorbance values at 222.5 and 241.5 nm versus the corresponding
concentrations, and then the regression equation was computed.
2.7.2. Analysis of Artificial Mixtures
Laboratory prepared mixtures containing DTA and different percentages
of its degradation product were prepared and analyzed using the same procedure
described under (Construction of Calibration Graphs).
2.7.3. Application of the Proposed Methods to the Analysis of DTA in
Pharmaceutical Preparation
Gastrografin® solution is labeled to contain 0.6 gm/mL of anhydrous
DTA. A stock solution with a concentration equal to 900 µg/mL was prepared
by transfer of 0.15 mL of the solution to a 100 mL volumetric flask and the
volume was completed using distilled water; from this stock solution, 0.1 mL
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was transferred into a 10 mL volumetric flask and the volume was completed
with distilled water to get a concentration equal to 9 µg/mL. Then the procedure
was completed as described under (Construction of Calibration Graphs).
3. Results and Discussion
DTA is partially deacetylated in liver to the deacetylated mutagenic and
cytotoxic metabolite 3,5-diamino-2,4,6 triiodobenzoate [15]. This free amino
compound can be also detected as an impurity in the final product as it is used as
a starting material for the synthesis of DTA which is formed by acetylation of
the 3,5-diamino-2,4,6 triiodobenzoate, so manufacturers of DTA should further
reduce the level of the aromatic amine [11]. This deacetylated degradate was
also obtained upon reflux of DTA with acid (Figure 1); therefore, the
determination of DTA in presence of its degradate was essential.
The structure of the degradate was elucidated by mass and IR
spectrometry in our previously published work [8].
The zero-order absorption spectra of DTA and its acidic degradate
showed that DTA acidic degradate overlaps with that of intact DTA and hinders
direct spectrophotometric measurements (Figure 2). The focus of the present
work is to develop accurate, specific, reproducible, and sensitive stability
indicating spectrophotometric methods for the determination of DTA in pure
form or in pharmaceutical formulations in the presence of its acidic degradation
product.
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3.1. Ratio Difference Method
Ratio difference method was recently developed as a new simple, rapid,
and selective method for the determination of components with overlapping
spectra, having the advantages of minimal data processing and wide range of
applications [16,17,18].
Ratio difference spectrophotometric method was applied to solve the
problem of the overlapped absorption spectra of DTA and its degradate as upon
dividing the spectrum of DTA by a divisor of a certain concentration of the
degradate, a ratio spectrum will result, and a linear relationship between the
difference in amplitudes at any two wavelengths and the corresponding
concentration of DTA will result, while the ratio spectrum of the degradate will
be a straight line of constant amplitude parallel to the x-axis and the difference
in amplitudes of it at any two wavelengths will be zero [19].
The method comprises two critical steps; the first is the choice of the
divisors; the selected divisors should compromise between minimal noise and
maximum sensitivity [20]. Different concentrations of the degradate were tried
2, 10, 14, and 20 µg/mL and the divisor concentration of 14 µg/mL was found to
be the best regarding sensitivity, repeatability, signal-to-noise ratio, and average
recovery percent when used for the prediction of DTA concentration in pure
powder form as well as in laboratory prepared mixtures (Figures 3 and 4). The
second critical step is the choice of the wavelengths at which the measurements
are recorded. Any two wavelengths can be chosen, provided that they exhibit
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different amplitudes in the ratio spectrum and a good linearity is present at each
wavelength individually. The wavelength pairs 220–238, 220–245, and 220–255
nm could not be used as 220 nm showed poor linearity. 243–250 and 243–253
nm were also tried and 243–253 nm showed the best results.
Linear correlation was obtained between the differences in amplitude at
243–253 nm for DTA in the range of 2–24 µg/mL, and the regression equations
were computed (Table 1). The methods were checked by the analysis of
laboratory prepared mixtures of DTA and its acidic degradate in different ratios
as presented in Table 2. DTA could be determined in presence of up to 90% of
its acidic degradate, with mean percentage recovery of 101.40 0.38%
3.2. Bivariate Method
This method is based on the simple mathematic algorithm [21], in which
data are used from four linear regression equations, two calibrations for each
component at two selected wavelengths using the method of Kaiser [22]. The
principle of bivariate calibration is in the measurements of binary mixtures (A
and B) at the two selected wavelengths (1 and 2), and then two equations are
obtained:
AAB1 = mA1CA + mB1CB + eAB1
AAB2 = mA2CA + mB2CB + eAB2
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where eAB1 and eAB2 are the sum of the intercepts of the linear calibration at two
wavelengths (eAB1 = eA1 + eB1), mA and mB are the slopes of the linear
regression, and CA and CB are the concentrations of the analytes.
The resolution of such equations set allows the evaluation of CA and CB
values:
CB = mA (AAB1 - eAB1) + mA1 (eAB2 - AAB2) / mA2mB1 - mA1mB2
CA = AAB1 - eAB1 - mB1CB / mA1
This simple mathematic algorithm allows the resolution of the binary
mixture by measuring the absorbance of the mixture at the two wavelengths and
using the parameters of the linear regression functions evaluated individually for
each component at these same wavelengths [23]. The method of Kaiser [22] was
used for the selection of optimum wavelength set which assured the best
sensitivity for the determination. A series of sensitivity matrices, K, was created
for each binary mixture and for every pair of preselected wavelengths:
K = mA1 mB1
mA2 mB2
where mA1,2 and mB1,2 are the slopes of the components A and B at the
two selected wavelengths (1, 2).
The determinants of these matrices were
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calculated and the wavelength set selected for which the highest matrix
determinant value was obtained.
Six wavelengths were taken and the slope values of the linear regression
equations were estimated for DTA and the degradate at the selected
wavelengths. Using the obtained data, the sensitivity matrices were created and
the respective determinants were calculated (Table 3).
For bivariate determination of DTA, the wavelengths 238 and 264 nm
were used. At these selected wavelengths, the calibration curves were obtained
in the range of 2–24 µg/mL and the linear regression equations were computed
(Table 1). DTA could be determined in presence of up to 90% of its acidic
degradate, with mean percentage recovery of 101.11 0.78% (Table 2).
3.3. Dual Wavelength Method
The developed dual wavelength method provides a simple method for
selective determination of DTA in the presence of its degradate using the zero-
order absorption spectra. The principle of this method is that the absorbance
difference at two points on the spectra is directly proportional to the component
of interest, independent of the interfering component [24]. The prerequisite for
this method is the selection of two wavelengths where the interfering component
shows the same absorbance value, while the component of interest shows
significant difference in absorbance with concentration [25]. Different pairs of
wavelengths were tried such as 242.6–290.3, 251.6–280.4, and 222.5–241.5 nm.
Using the absorbance values at 222.5 and 241.5 nm gave the best selectivity.
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Linear correlation was obtained between the differences absorbance
values at the selected wavelengths in amplitude at 222.5–241.5 nm for DTA in
the range of 2–24 µg/mL, and the regression equations were computed (Table
1). The methods were checked by the analysis of laboratory prepared mixtures
of DTA and its acidic degradate in different ratios as presented in Table 2. DTA
could be determined in presence of up to 90% of its acidic degradate, with mean
percentage recovery of 99.77 0.73%.
The usefulness of the proposed methods was successfully applied to the
analysis DTA in its pharmaceutical formulation and was studied in presence of
additives by assaying Gastrografin® solution. The validity of the methods was
assessed by applying the standard addition technique (Table 4).
Results obtained by the proposed procedures for the determination of
pure samples of the drug were statistically compared to those obtained by the
official US pharmacopeial method and no significant difference was observed
(Table 5). The official method depends on using standard 0.05N AgNO3 as a
titrant and tetrabromophenolphthalein as an indicator [14]. Method validation
was performed according to the USP guidelines [14] for all the proposed
methods. Table 1 shows results of accuracy, repeatability, and intermediate
precision of the methods.
The three methods described have a common feature of choosing two
wavelengths. Unlike the other two methods, the ratio difference method allows
the selection of any two wavelengths for determination of the drug, such as
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wavelengths of maximum absorbance that can increase the sensitivity of the
method. In bivariate method, upon applying Kaiser method [21] for selecting the
wavelengths for bivariate method, wavelengths that has low sensitivity may be
introduced as shown in the regression equation for DTA at 264 nm. The dual
wavelength method has a main disadvantage that the selected wavelengths often
meet at peak shoulders, where ∆A/∆λ is said to be maximum; therefore the
selectivity towards the measured component is extremely affected. Also in high
concentrations in the synthetic mixtures, there is slight change in the absorption
spectrum of each component which may affect the subtraction step and the
obtained recoveries, while in ratio difference method measurements can be done
at any two wavelengths throughout the whole ratio spectrum including minima
or maxima. These limitations may show the superiority of ratio difference
method over the other two methods. The ratio difference method also has
advantages over methods manipulating ratio spectra in being very simple (one
step method).
Conclusion
The three described spectrophotometric methods have the advantages of
being more simple and can analyze DTA in its pure powder and dosage form in
the presence of up to 90% of its cytotoxic degradation product without complex
calculations or multistep procedures. Unlike the chromatographic methods, these
methods do not require expensive solvents, sample preparation, buffers, or
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sophisticated techniques and instruments. The methods are suitable and valid for
application in laboratories lacking liquid chromatographic instruments. High
values of correlation coefficients and small values of intercepts validated the
linearity of the calibration graphs and the validity of Beer’s law. The new and
simple ratio difference method shows some advantages over the bivariate and
dual wavelength methods. From the results obtained, we concluded that the
suggested methods showed high sensitivity, accuracy, reproducibility, and
specificity and can be used as stability indicating methods in quality control
laboratories.
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Acidic degradation
+ 2 CH3COOH
Sodium diatrizoate
3,5 diamino-2,4,6 triiodobenzoic acid
Figure 1: The suggested mechanism of degradation of DTA.

Abs
orb
anc
e
Wavelength (nm)
Figure 2: Zero order absorption spectra of DTA 14 µg/mL (
degradate 14 µg/mL(-----).
) and the

Figure 3: Ratio spectra of DTA (2 - 24 µg/mL) using the spectrum of
degradate (14 µg/mL) as a divisor.

Figure 4 : Ratio spectra of DTA 10 µg/mL (- - - ), degradate 10 µg/mL (
) , and a laboratory
ƉƌĞƉĂƌĞĚꢀŵŝdžƚƵƌĞꢀĐŽŶƚĂŝŶŝŶŐꢀϭϬꢀђŐͬŵ>ꢀŽĨꢀďŽƚŚꢀ;͙͙͘ͿꢀƵƐŝŶŐꢀĂꢀĚŝǀŝƐŽƌꢀŽĨꢀϭϬꢀђŐͬŵ>ꢀĚĞŐƌĂĚĂƚĞ͘

Table 1: Assay validation sheet of the proposed methods for the determination of pure DTA
Ratio Difference
method
Dual Wavelength
method
parameter
Bivariate method
Accuracy
(mean SD)
Repeatability a
Intermediate
precision b
Specificity
Linearity
Slope
100.63 1.06
0.954
100.02 0.868
1.050
100.06 0.754
0.770
0.912
0.415
0.680
101.40 0.389
101.11 0.787
99.77 0.735
0.0533
0.0070
0.0467
0.0116
0.0130
0.0037
Intercept
r2
0.9996
0.9993
0.9999
Range
2 - 24 µg/mL
2 - 24 µg/mL
2 - 24 µg/mL
aThe intraday and b the inter-day RSD values of samples of concentration of 7,11 and 17 µg/mL of DTA.

Table 2 : Determination of DTA in laboratory-prepared mixtures by the proposed methods
Dual
% degradation
product
Ratio Difference
method
Bivariate
method
Concentration (µg/mL)
Wavelength
method
10
20
18
16
14
12
10
8
2
4
101.89
101.42
100.94
101.65
101.32
101.57
100.67
101.77
101.36
101.40
0.389
101.96
100.18
101.12
99.56
98.85
99.42
99.62
99.71
101.31
99.81
100.38
99.86
99.02
99.77
0.735
0.736
30
6
40
8
50
10
12
14
16
18
101.05
101.68
101.14
101.51
101.79
101.11
0.787
60
70
6
80
4
90
2
mean
SD
RSD%
0.383
0.778

Table 3: Application of Kaiser method for wavelength selection (K* 10-6)
ȜꢀꢁꢀȜ
230
238
246
252
264
270
230
0
238
959.2
0
246
1699
2032
0
252
2427
2903
2391
0
270
264
3371
4031
3320
2651
0
3243
3879
3194
2550
66.56
0

Table 4: Quantitative determination of DTA in Gastrografin® solution by the proposed
methods and the results of application of standard addition technique
Ratio Difference
method
Bivariate
method
Dual Wavelength
method
Gastrografin® solution
Recovery SD a
100.70 1.01
Batch no.51424A
100.66 0.44
99.49 0.93
100.60 0.67
100.44 0.62
Recovery% of standard added
100.44 0.96
aAverage of three determinations.
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Table 5: Statistical analysis of the results obtained by the proposed methods and the
official method for the determination of DTA in pure powder form
Ratio
Difference
method
Dual Wavelength
Item
Bivariate method
Official method a
method
100.06
0.75
Mean
SD
100.63
1.06
1.12
12
100.20
0.87
0.76
12
100.85
0.97
0.94
5
Variance
n
0.56
12
Student's t-test b
0.399 (2.13)
1.19 (5.41)
1.36 (2.13)
1.23 (2.98)
1.82 (2.13)
1.67 (2.98)
F-value b
a
Precipitimetric titration using standard 0.05N AgNO3 and tetrabromophenolphthalein
indicator.
b Figures between parentheses represent the corresponding tabulated values of t and F at
P = 0.05.

Deg.
DTA
241.5
222.5
Sodium diatrizoate
Ac
idi
c
de
gr
ad
ati
on
+ 2 CH3COOH
3,5 diamino-2,4,6 triiodobenzoic acid
ȜꢀꢁꢀȜ 230 238
246 252
270
264
230
0
959.2 1699 2427 3371 3243
2903
0
2032
0
3879
4031
238
246
252
264
270
2391 3320 3194
2651 2550
0
0
66.56
0

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• A comparative study involving three stability indicating
spectrophotometric methods.
• The three methods are based on measuring at two wavelengths.
• No need for sophisticated instruments or preliminary separation steps.
• Simple, inexpensive, and applicable methods for quality control
laboratories.
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