Stability indicating spectrophotometric and spectrodensitometric methods for the determination of diatrizoate sodium in presence of its degradation product

Article abstract of DOI:10.1016/j.saa.2014.10.002

Three sensitive, selective, and precise stability indicating 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 a first derivative (D₁) spectrophotometric one, which allows the determination of DTA in the presence of its degradate at 231.2 nm (corresponding to zero crossing of the degradate) over a concentration range of 2-24 μg/mL with mean percentage recovery 99.95 ± 0.97%. The second method is the first derivative of the ratio spectra (DD₁) by measuring the peak amplitude at 227 nm over the same concentration range as D₁ spectrophotometric method, with mean percentage recovery 99.99 ± 1.15%. The third method is a TLC-densitometric one, where DTA was separated from its degradate on silica gel plates using chloroform:methanol:ammonium hydroxide (20:10:2 by volume) as a developing system. This method depends on quantitative densitometric evaluation of thin layer chromatogram of DTA at 238 nm over a concentration range of 4-20 μg/spot, with mean percentage recovery 99.88 ± 0.89%. 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.2014.10.002

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 136 (2015) 1167–1174
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Stability indicating spectrophotometric and spectrodensitometric
methods for the determination of diatrizoate sodium in presence of its
degradation product
⇑
Mohamed K. Abd El-Rahman, Safaa M. Riad, Sherif A. Abdel Gawad, Esraa M. Fawaz , Mostafa A. Shehata
Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, Kasr-El Aini Street, 11562 Cairo, Egypt
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
ꢀ
First stability indicating
spectrophotometric and TLC methods
for DTA.
Deg.
DTA
ꢀ
ꢀ
ꢀ
No need for a preliminary separation
step.
Sodium diatrizoate
First degradation study involving the
cytotoxic diamino degradate.
Simple, inexpensive, and applicable
methods for quality control
laboratories.
+ 2 CH3COOH
3,5 diamino-2,4,6 triiodobenzoic acid
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 12 July 2014
Received in revised form 9 September 2014
Accepted 4 October 2014
Available online 16 October 2014
Three sensitive, selective, and precise stability indicating methods for the determination of the X-ray con-
trast 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 a first derivative (D1) spectrophotometric one, which allows the determination of DTA in
the presence of its degradate at 231.2 nm (corresponding to zero crossing of the degradate) over a con-
centration range of 2–24 lg/mL with mean percentage recovery 99.95 0.97%. The second method is the
Keywords:
Sodium diatrizoate
Derivative spectrophotometry
TLC-densitometry
Stability indicating methods
first derivative of the ratio spectra (DD1) by measuring the peak amplitude at 227 nm over the same con-
centration range as D1 spectrophotometric method, with mean percentage recovery 99.99 1.15%. The
third method is a TLC-densitometric one, where DTA was separated from its degradate on silica gel plates
using chloroform:methanol:ammonium hydroxide (20:10:2 by volume) as a developing system. This
method depends on quantitative densitometric evaluation of thin layer chromatogram of DTA at
238 nm over a concentration range of 4–20 lg/spot, with mean percentage recovery 99.88 0.89%. The
selectivity of the proposed methods was tested using laboratory-prepared mixtures. The proposed meth-
ods have been successfully applied to the analysis of DTA in pharmaceutical dosage forms without inter-
ference 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.
Ó 2014 Elsevier B.V. All rights reserved.
Introduction
X-ray diagnostic agents (contrast media) are widely used as
adjuncts to diagnostic visualization techniques as they help to
⇑
Corresponding author. Tel.: +20 1005639834.
E-mail address: esraafawaz_2007@yahoo.com (E.M. Fawaz).
http://dx.doi.org/10.1016/j.saa.2014.10.002
1386-1425/Ó 2014 Elsevier B.V. All rights reserved.

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M.K. Abd El-Rahman et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 136 (2015) 1167–1174
illustrate the differences between tissues by introducing them to
the area of interest to increase its density and absorption of X-rays;
thus, they can enhance the image obtained. Radiographic contrast
media are based on elements with high atomic numbers that can
absorb X-rays. The most commonly used contrast media are the
iodinated organic compounds, particularly tri-iodinated benzene
compounds, and their degree of opacity or radiodensity is directly
proportional to their iodine content [1–3]. Iodine-based contrast
agents are classified according to their structures into ionic and
nonionic, and diatrizoate sodium (DTA) is one of the most widely
used ionic contrast agents in many medical imaging purposes
either intravenously, such as angiography, pyelography, computed
tomography, and myelography, or orally or rectally as enema in
gastrointestinal imaging [4].
Pharmaceutical formulation
GastrografinÒ solution manufactured by Schering Company
(Belimed, Spain), batch number 51424A, labeled to contain 0.6 g/
mL of diatrizoate anhydrous base.
Degraded samples
10 mL of 2 M HCl solution was added to pure DTA (500 mg) in a
250 mL glass-stoppered conical flask and refluxed for 6 h. Com-
plete 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. Subse-
quently, 2 M NaOH solution was added to the degraded solution till
pH was adjusted to about 7, then the solution was evaporated on
small flame, and finally the degradate is dissolved in methanol, fil-
tered, and left to evaporate at room temperature. The structure of
the isolated degradation product was elucidated using mass and IR
spectrometry.
In modern analytical laboratory, there is always a need for sig-
nificant stability indicating methods of analysis [5,6]. An ideal sta-
bility indicating method quantifies
a drug and resolves its
degradation products [7]. Reviewing the literature in hand
revealed that there is no stability indicating method for the deter-
mination of DTA in presence of its free amino degradate in spite of
its high toxicity. There are only one direct UV spectrophotometric
method [8] and another H1NMR method for the determination of
DTA as a single component [9]. DTA was also determined in pres-
ence of its di-iodo degradates by capillary electrophoresis tech-
nique [10] and liquid chromatography [11,12]. DTA is an official
drug whose US pharmacopeial assay method is a precipitimetric
titration that depends on the iodide content in the drug [13].
The aim of the present work was to develop and validate stabil-
ity indicating methods for the determination of DTA in presence of
its acidic 3,5-diamino degradate which also can be formed in vivo
by deacetylation via liver microsomal enzymes. It is proved that it
has a mutagenic and cytotoxic effect on the body [14]. The pro-
posed methods are applicable to the quality control and routine
analysis of DTA in pharmaceutical preparations. These methods
include first derivative (D1), first derivative of the ratio spectra
(DD1), and TLC-densitometry.
Materials and reagents
All chemicals used throughout this work were of analytical
grade, and the solvents were of spectroscopic grade. Methanol
and chloroform were purchased from VWR International (West
Chester, PA).
Standard solutions
DTA stock standard solutions with a concentration of 0.1 mg/mL
in distilled water and 1 mg/mL in methanol for the spectrophoto-
metric and TLC-densitometric methods, respectively, were pre-
pared in 100 mL volumetric flasks by dissolving 10 and 100 mg
of pure DTA in distilled water and methanol. Degradation product
stock solutions with a concentration of 0.1 mg/mL in distilled
water for the spectrophotometric methods and 1 mg/mL in meth-
anol for the TLC-densitometric method were prepared.
Experimental
Procedures
Apparatus
Construction of calibration graphs
Spectrophotometer: SHIMADZU dual beam (Kyoto/Japan) UV–
visible spectrophotometer model UV-1601 PC. TLC plates pre-
coated with silica gel F254, 0.25 mm thickness (Merck, Darmstadt,
Germany). TLC scanner 3 densitometer model 3 S/N 130319
(CAMAG, Muttenz, Switzerland). Camag Linomat 5 autosampler
D1 method. 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 water. The zero-
order spectra were recorded using distilled water as a blank. The
first derivative of the obtained spectra was recorded using
with Camag microsyringe (100 lL); (CAMAG, Muttenz, Switzer-
D
k = 4 nm and the scaling factor 50. The peak amplitudes of the
land). Camag TLC scanner – Model 3 S/N 130319 with winCats soft-
ware (CAMAG, Muttenz, Switzerland).
obtained first derivative spectra were measured at 231.2 nm. Cali-
bration graph relating the peak amplitude to the corresponding
concentrations of DTA was constructed, and the corresponding
regression equation was computed.
Reference samples
Diatrizoate sodium reference standard was purchased from
DD1 method. Aliquots (0.2–2.4 mL) of DTA stock solution (0.1 mg/
Sigma–Aldrich Co., and its purity was certified to be P99.99%.
mL) were transferred into a series of 10 mL volumetric flasks,
+ 2 CH3COOH
Sodium diatrizoate
3,5 diamino-2,4,6 triiodobenzoic acid
Fig. 1. The suggested mechanism of degradation of DTA.

M.K. Abd El-Rahman et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 136 (2015) 1167–1174
1169
Fig. 2. IR spectrum of intact DTA.
Forked NH2
Fig. 3. IR spectrum of DTA degradation product.
and the volume was completed with distilled water. The zero-
order spectra of the prepared solutions were divided by the spec-
and 15 mm from the bottom edge of the plate with a band length
of 6 mm. The plates were developed in chromatographic tanks pre-
viously saturated with the mobile phase chloroform: methanol:
ammonium hydroxide (20:10:2 by volume), by ascending chroma-
tography. The plates were dried, spots were detected under the UV
lamp (254 nm), and the plates were scanned at 238 nm. A calibra-
tion graph relating the peak to the corresponding concentration of
DTA was constructed, and the regression equation was computed.
trum of 14
tive of the ratio spectra (DD1) was obtained using scaling factor
50 and k = 4 nm. The peak amplitudes of the first derivative of
lg/mL degradation product, and then the first deriva-
D
the ratio spectra were measured at 227 nm. Calibration graph
relating the peak amplitudes of (1DD227) to the corresponding con-
centrations of DTA was constructed, and the corresponding regres-
sion equation was computed.
TLC-densitometric method. Aliquots (4–20
l
L) from DTA stock solu-
Analysis of artificial mixtures
tion (1.0 mg/mL) prepared in methanol were spotted onto a TLC
plate using Camag Linomat autosampler with Camage microsy-
ringe (100 lL). Spots were spaced 10.5 mm apart from each other
Laboratory-prepared mixtures containing DTA and different
percentages of its degradation product were prepared and ana-
lyzed by the proposed methods.

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Wavelength (nm)
Fig. 4. Zero order absorption spectra of DTA 14 lg/ml (—) and the degradate 14 lg/
ml (-----).
Fig. 6. First derivative absorption spectra of DTA (2–24 lg/ml).
Wavelength (nm)
Fig. 5. First derivative absorption spectra of DTA 14
14 g/ml (-----).
lg/ml (—) and the degradate
l
Application of the proposed methods to the analysis of DTA in
pharmaceutical preparation
Fig. 7. Ratio spectra of DTA (2–24 lg/ml) using the spectrum of degradate (14 lg/
ml) as a divisor.
D1 and DD1 methods. GastrografinÒ solution is labeled to contain
0.6 gm/mL of anhydrous DTA. A stock solution with a concentra-
tion equal to 900 lg/mL was prepared by transfer of 0.15 mL of
the solution to a 100 mL volumetric flask and the volume was com-
pleted using distilled water; from this stock solution 0.1 mL was
transferred into a 10 mL volumetric flask and the volume was com-
Results and discussion
pleted with distilled water to get a concentration equal to 9 lg/mL.
Then the procedure was completed as described in Sections ‘D1
method and DD1 method’ of construction of calibration graphs.
DTA is partially deacetylated in liver to the deacetylated muta-
genic and cytotoxic metabolite 3,5-diamino-2,4,6 triiodobenzoate
[10]. This free amino compound can be also detected as an impu-
rity 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-dia-
mino-2,4,6 triiodobenzoate; therefore, manufacturers of DTA
should further reduce the level of the aromatic amine [10]. This
deacetylated degradate was also obtained upon reflux of DTA with
acid or alkali (Fig. 1); therefore, the determination of DTA in pres-
ence of its degradate was essential.
TLC-densitometric method. From GastrografinÒ solution 0.1 mL was
transferred to 100 mL volumetric flask and the volume was com-
pleted with methanol. 10 lL of the prepared solution was spotted
in triplicate using Linomat applicator onto a TLC plate, and the pro-
cedure was completed as described in Section ‘TLC-densitometric
method’ of construction of calibration graphs.

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1171
The focus of the present work was to develop accurate, specific,
reproducible, and sensitive stability indicating methods for the
determination of DTA in pure form or in pharmaceutical formula-
tions in the presence of its acidic degradation product.
D1 and DD1 methods
The zero-order absorption spectra of DTA and its acidic degra-
date showed that DTA acidic degradate overlaps with that of intact
DTA and hinders direct spectrophotometric measurements (Fig. 4).
Derivative spectrophotometry is an analytical technique of great
utility for extracting both qualitative and quantitative information
from spectra composed of unresolved bands and for eliminating
the effect of baseline shifts and baseline tilts by using the first or
higher derivatives of absorbance with respect to wavelength [15].
A rapid, simple, and low cost spectrophotometric method based
on measuring the peak amplitude of D1 spectrum of DTA at
231.2 nm (corresponding to zero crossing of the degradate) was
developed with good selectivity without interference of the degra-
date as shown in Figs. 5 and 6. In order to optimize D1 method, dif-
ferent smoothing and scaling factors were tested, where
smoothing factor k = 4 and a scaling factor = 50 showed a suitable
signal to noise ratio and the spectra showed good resolution.
a
D
Fig. 8. First derivative of ratio spectra of DTA (2–24
degradate (14 g/ml) as a divisor.
l
g/ml) using the spectrum of
In order to improve the selectivity of the analysis of DTA in
presence of its acidic degradate, DD1 spectrophotometric method
was also established. The principal advantage of the method is that
the whole spectrum of interfering substance is cancelled, so the
choice of the wavelength used for calibration is not critical as in
the D1 method [16,17].
l
The structure of the degradate was elucidated by mass spec-
trometry, and the electron ionization showed mass ion peak at
m/z 521 corresponding to the acidic 3,5-diamino degradate as
shown in Fig. S-1 (Supplementary materials). Moreover, the struc-
ture was further confirmed by IR spectrometry which indicated
two forked peaks corresponding to the two primary amino groups
at 3460 and 3381 cmÀ1 (Figs. 2 and 3).
In order to optimize DD1 method, several divisor concentrations
2, 8, and 14
was obtained when using 14
Different k and scaling factors were tested, where
scaling factor = 50 were suitable to enlarge the signal of DTA to
l
g/mL of the degradate were tried, and the best result
g/mL of the degradate as a divisor.
k = 4 and a
l
D
D
Table 1
Assay validation sheet of the proposed methods for the determination of pure DTA.
Parameter
D1 method
DD1 method
TLC-densitometric method
Accuracy (mean SD)
Repeatabilitya
99.95 0.97
0.643
1.290
99.99 1.15
0.682
1.030
99.88 0.89
0.371
0.990
Intermediate precisionb
Specificity
100.95 1.37
100.94 0.81
101.02 1.19
Linearity
Slope
0.0624
0.0072
0.9998
0.5169
0.0479
0.9993
0.0493
0.3050
0.9997
Intercept
r2
Range
2–24
lg/ml
2–24
lg/ml
4–20 lg/spot
a
The intraday RSD values of samples of concentration of 7, 11 and 17
The inter-day RSD values of samples of concentration of 7, 11 and 17
l
l
g/ml of DTA for D1 and DD1, and 5, 11 and 17 g/spot for TLC.
g/ml of DTA for D1 and DD1, and 5, 11 and 17 lg/spot for TLC.
l
b
Table 2
Determination of DTA in laboratory-prepared mixtures by the proposed methods.
% Degradation product
Concentration (
l
g/mL for D1 and DD1) and (
l
g/spot for TLC)
D1 method
DD1 method
TLC-densitometry
10
20
30
40
50
60
70
80
18
16
14
12
10
8
6
4
2
2
4
6
101.12
98.64
98.61
99.11
99.04
101.93
100.97
99.58
100.87
101.62
101.69
101.87
100.74
1.19
101.00
101.64
100.70
102.07
98.53
8
101.00
101.63
101.92
101.67
101.98
102.05
100.96
1.37
10
12
14
16
18
101.59
101.63
90
Mean
SD
101.02
1.19
RSD%
1.36
1.18
1.18

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Retenꢀon ꢀme (minutes)
Fig. 9. 2D TLC chromatogram of DTA (Rf = 0.4) and its degradate (Rf = 0.22).
Fig. 10. 3D TLC chromatogram of DTA (4–20 lg/ml).
facilitate its measurement and to diminish error in reading the sig-
nal (Figs. 7 and 8). The absorption spectra of DTA in the range of 2–
24 lg/mL were divided by the absorption spectrum of 14 lg/mL of
The linearity of the peak amplitudes of the D1 curves at
231.2 nm and the peak amplitudes of the DD1 curves at 227 nm
was studied. A linear relationship was obtained in the range of
the degradate (as a divisor), where the obtained ratio spectra were
differentiated with respect to wavelength. DD1 values showed
good linearity and reproducibility at 227 nm.
2–24 lg/mL for the drug, and the regression equations were com-
puted (Table 1). The methods were checked by analysis of labora-
tory-prepared mixtures of DTA and its acidic degradate in different

M.K. Abd El-Rahman et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 136 (2015) 1167–1174
1173
Table 3
Quantitative determination of DTA in GastrografinÒ solution by the proposed methods and the results of application of standard addition technique.
GastrografinÒ solution
D1 method
DD1 method
TLC-densitometry
Recovery SDa
Batch no.51424A
Recovery % of standard added
101.51 0.98
101.11 0.41
100.43 1.23
100.27 1.08
100.54 0.89
100.59 0.84
a
Average of three determinations.
Table 4
Statistical analysis of the results obtained by the proposed methods and the official method for the determination of DTA in pure powder form.
Item
D1 method
DD1 method
TLC-densitometric method
Official methoda
Mean
SD
99.95
0.97
0.94
12
1.74 (2.13)
1.00 (5.41)
99.99
1.15
1.32
12
1.46 (2.13)
1.43 (5.41)
99.88
0.89
0.79
9
1.89 (2.17)
1.19 (3.84)
100.85
0.97
0.94
5
Variance
n
Student’s t-testb
F-valueb
a
Precipitimetric titration using standard 0.05 N AgNO3 and tetrabromophenolphthalein indicator.
Figures between parentheses represent the corresponding tabulated values of t and F at P = 0.05.
b
ratios as presented in Table 2. DTA could be determined in pres-
ence of up to 90% of its acidic degradate in case of D1 and DD1
methods, with mean percentage recovery of 100.95 1.37% and
100.78 1.23%, respectively.
Comparing the two proposed spectrophotometric methods, DD1
method shows more advantages over D1 method as it avoids criti-
cal measurements at zero crossing points where 1 nm shift
between different spectrophotometers in different laboratories
can seriously affect the results of D1 method; thus, DD1 method
reveals better accuracy and method transfer.
The usefulness of the proposed methods was successfully
applied to the analysis of DTA in its pharmaceutical formulation
and was studied in presence of additives by assaying GastrografinÒ
solution. The validity of the methods was assessed by applying the
standard addition technique (Table 3).
The advantage of the TLC-densitometric method is that it is the
easiest and fastest way not only for quantitation of the drug but also
for visual detection of impurities or trace amounts of the toxic
degradate which cannot be easily detected by the UV-spectrophoto-
metric methods. Moreover, it is simple and inexpensive permitting
its application in quality control laboratories where spectrophoto-
metric derivatization programs are not applicable, also it avoids fur-
ther data manipulation and derivatization steps.
TLC-densitometric method
Results obtained by the proposed procedures for the determina-
tion of pure samples of the drug were statistically compared to
those obtained by the US pharmacopeial method and no significant
difference was observed (Table 4). The official method depends on
using standard 0.05 N AgNO3 as a titrant and tetrabromophenolph-
thalein as an indicator [13]. Method validation was performed
according to USP guidelines [18] for all the proposed methods.
Table 1 shows results of accuracy, repeatability, and intermediate
precision of the methods.
A stability indicating TLC-densitometric method for the deter-
mination of DTA was also described. Several trials were done to
choose a developing system which can separate DTA from its deg-
radation product including methanol: ethyl acetate: ammonium
hydroxide (10:5:0.1 by volume), methanol: chloroform: ammo-
nium hydroxide (5:5:0.1 by volume), and chloroform: methanol:
ammonium hydroxide (10:5:1 by volume). The first and the second
systems were not satisfactory because they did not affect good sep-
aration of the drug from its degradation product, the first system
showed no separation, while the second one separated the two
spots but with Rf close to each other (Rf = 0.18 and 0.15 for intact
drug and degradation product, respectively). Satisfactory separa-
tion was obtained using the third system, chloroform: methanol:
ammonium hydroxide (10:5:1 by volume) as a mobile phase.
Respective Rf values were 0.4 0.02, 0.22 0.02 for DTA and its
degradate, respectively, as shown in Fig. 9. This separation allowed
the determination of DTA at 238 nm without any interference from
the degradation product as shown in Figs. 10 and S-4 (Supplemen-
tary materials).
Conclusion
In the present work, three simple, sensitive, and rapid methods
are described for determination of DTA in pure form or in pharma-
ceutical formulations. The proposed D1 and DD1 spectrophotomet-
ric methods are simple, more convenient, less time consuming, and
more economic stability indicating methods compared to other
published methods. The proposed methods have the advantage
of being applicable in the presence of up to 90% of the degradate
for the spectrophotometric methods and up to 80% for TLC-densi-
tometric method. The advantages of TLC-densitometric method are
that several samples can be run simultaneously using a small
quantity of mobile phase unlike HPLC, thus lowering analysis time
and cost per analysis, and provide high sensitivity and selectivity.
High values of correlation coefficients and small values of inter-
cepts validated the linearity of the calibration graphs and the valid-
ity of Beer’s law. From the results obtained, we concluded that the
suggested methods showed high sensitivity, accuracy, reproduc-
ibility, and specificity and can be used as stability indicating meth-
Linear relationship was found to exist between the area ratio
calculated by division of the peak area of each concentration by
the peak area of a certain chosen concentration (14
is spotted on the same plate and the corresponding concentration
of DTA in the range of 4–20 g/spot at the selected wavelength
lg/spot) which
l
(238 nm). The regression equation was computed (Table 1). The
proposed method was valid for the determination of DTA in differ-
ent laboratory-prepared mixtures in presence of up to 80% of its
acidic degradate (Table 2), with mean percentage recovery of
101.02 1.19%.

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ting their application in quality control laboratories.
Appendix A. Supplementary material
[12] E.M. Chellquist, W.O. Nelson, H.L. Storflor, J. Pharm. Biomed. Anal. 16 (1)
(1997) 39–45.
Supplementary data associated with this article can be found, in
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[13] The United States Pharmacopeia and National Formulary, The Official
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