Selective and Validated Spectrophotometric Assay for Microgram Determination of Ganciclovir with 1-fluoro-2, 4-dinitrobenzene and N-Bromosuccinimide Reagents

Abstract

Two simple, sensitive, selective, accurate, precise and economical methods (method A and B) have
been developed for the quantitative estimation of ganciclovir in bulk drug and its pharmaceutical
formulations. In method A, an aqueous solution of ganciclovir reacts with 1-fluoro-2, 4-dinitrobenzene
(Sanger’s reagent) at borate buffer pH 9 and forms a yellow color complex and absorbance was
measured at 354 nm. In method B, N-bromosuccinimide (NBS) used as an oxidimetric reagent and
dye methyl orange was used as a spectrophotometric reagent. The method involves adding a measured
excess of NBS to ganciclovir in acid medium followed by determination of residual NBS by reacting
with a fixed amount of methyl orange and measuring the absorbance at 508 nm. The Beer’s law
was obeyed in the concentration range of 0.2-0.6 µg/ml, 1-5μg/ml for method A and B respectively.
The accuracy and reliability of the methods were further ascertained by performing recovery tests
via standard-addition method. The recoveries of ganciclovir tablets are in the range 99.24, 99.16
respectively. The proposed method is simple, rapid, precise and convenient for the assay of ganciclovir
in commercial tablet preparations.

Full text

Journal of Applied Chemical Research, 20, 1, 14-27 (2012)
Journal of
Applied
Chemical
Research
www.jacr.kiau.ac.ir
* Corresponding auther: Dr. BM Gurupadayya, Department of Pharmaceutical Analysis, JSS College of Pharmacy, JSS Uni-
versity, Shivarathreashwara Nagar, Mysore-570 015, Karnataka, India, Fax Number: +91-821-2548359, Mobile Number: +91-
9242886136, E mail: bm_guru2004@yahoo.co.in
Selective and Validated Spectrophotometric Assay for
Microgram Determination of Ganciclovir with 1-uoro-2,
4-dinitrobenzene and N-Bromosuccinimide Reagents
T. Anil Kumar, B. M. Gurupadayya*, M.B. Rahul Reddy
Department of Pharmaceutical Analysis, JSS College of Pharmacy, JSS University, Mysore, India
(Received 05 June 2011; Final version received 15 November 2011)
Abstract
Two simple, sensitive, selective, accurate, precise and economical methods (method A and B) have
been developed for the quantitative estimation of ganciclovir in bulk drug and its pharmaceutical
formulations. In method A, an aqueous solution of ganciclovir reacts with 1-uoro-2, 4-dinitrobenzene
(Sanger’s reagent) at borate buffer pH 9 and forms a yellow color complex and absorbance was
measured at 354 nm. In method B, N-bromosuccinimide (NBS) used as an oxidimetric reagent and
dye methyl orange was used as a spectrophotometric reagent. The method involves adding a measured
excess of NBS to ganciclovir in acid medium followed by determination of residual NBS by reacting
with a xed amount of methyl orange and measuring the absorbance at 508 nm. The Beer’s law
was obeyed in the concentration range of 0.2-0.6 µg/ml, 1-5μg/ml for method A and B respectively.
The accuracy and reliability of the methods were further ascertained by performing recovery tests
via standard-addition method. The recoveries of ganciclovir tablets are in the range 99.24, 99.16
respectively. The proposed method is simple, rapid, precise and convenient for the assay of ganciclovir
in commercial tablet preparations.
Keywords: Ganciclovir (95.3%), 1-uoro-2, 4-dinitrobenzene (Sanger’s reagent), N-Bromosuccinimide
(NBS)
Introduction
Ganciclovir (GCV) is chemically 2-amino-
1,9-[{2-hydroxyl-1-(hydroxymethyl)ethoxy}
methyl]-6H-purine-6H-one. Ganciclovir is
an acyclic guanosine analog that requires
triphosphorylation for activation prior to
inhibiting the viral DNA polymerase. It is
used in treatment of cytomegalovirus (CMV)
infection in AIDS patients [1]. Ganciclovir
exhibit antiviral activity against herpes simplex
virus (HSV) and cytomegalovirus (CMV) at
relatively low inhibitory concentrations. It is
ofcial in Martindale [2] Merck Index [3] and
USP [4]. Literature survey reveals that few

T. Anil Kumar et al., J. Appl. Chem. Res., 20, 1, 14-27 (2012) 15
methods like liquid chromatography using
pulsed amperometric detection in plasma [5],
high performance liquid chromatography
(HPLC) with pre column uorescence deviation
using phenyl glyoxal in serum [6].
Several methods which include
radioimmunoassay (RIA) [7] and enzyme-
linked immunosorbent assay [8, 9] have been
initially reported for the quantication of this
GCV in biological uids. Some combination
method for simultaneous determination
of GCV, and acyclovir by ow-injection
chemiluminescence method [10], acyclovir
and penciclovir in human plasma using
uorescence detection [11], teicoplanin in
plasma [12] and acyclovir and guanine [13]
were developed. Only few spectrophotometric
methods [14,15] are reported for the estimation
of ganciclovir using spectrophotometry in
bulk drug or its formulations.
Sanger’s reagent has been utilized as a chromogen
for the spectrophotometric estimation of many
compounds of pharmaceutical interest such as
desloratadine [16], enalapril [17], lisinopril [18]
and gabapentin [19]. A great number of organic
compounds have been spectrophotometrically
determined using excess NBS as oxidant in the
presence of celestine blue for propranolol and
tetracycline hydrochlorides [20], omeprazole
[21] azathioprine and astemizole [22]. However,
the reactions of N-Bromo succinamide
with ganciclovir and Sanger’s reagent with
ganciclovir have not been investigated so far.
The present study describes the evaluation of
N-Bromo succinamide and Sanger’s reagent
used as reagents for the development of simple
and rapid spectrophotometric method for the
determination ganciclovir in its pharmaceutical
dosage forms.
Method & Materials
Instruments
A double-beam Shimadzu 1700 UV
spectrophotometer, connected to computer and
loaded with UV solution software was used.
For an intermediate precision study and for
ruggedness, a different Shimadzu 1800 UV
spectrophotometer connected to computer with
UV-PC software was used. Both instruments
have an automatic wavelength accuracy of
0.1 nm and matched quartz cells of 10 mm
(1.0 cm) cell path length. The absorbance of
ganciclovir in the selected medium at respective
wavelength was determined and the apparent
molar absorptivity was calculated.
1-Flouro-2-4-dinitro benzene (Sanger’s
reagent) 0.5 %( w/v)
0.5 g of Sanger’s reagent was accurately
weighed transferred into a 100 ml calibrated
ask, dissolved in methanol, and make up the
volume up to the mark to obtain a solution
of 0.5% (w/v). Reagent should be protected
from light during use and should be handled
carefully since it is a skin irritant. It is stored
in a refrigerator and it was stable for 4 months.

T. Anil Kumar et al., J. Appl. Chem. Res., 20, 1, 14-27 (2012)
16
Borate buffer pH 9
Place 50ml 0.2M boric acid, 50 ml 0.2M
potassium chloride into 200ml volumetric
ask, then add 20 ml of 0.2M NaOH and nally
make up the volume with distilled water.
N-Bromosuccinimide (NBS) 0.02 %( w/v)
0.02 g of N-Bromosuccinimide was accurately
weighed transferred into a 100 ml calibrated
ask and make up the volume up to the mark
with distilled water. The solution was freshly
prepared and protected from light during the use.
Methyl orange 0.01%
0.01 g of methyl orange is accurately weighed
and transferred into a 100.0ml volumetric ask
and dissolved it with small quantity of water,
then made up to the mark with distilled water.
Hydrochloric acid 1M
8.5 ml of concentrated HCl is accurately measured
and transferred into a 100.0 ml volumetric ask
and made up to the mark with distilled water.
Preparation of standard solution
Accurately weighed 100 mg of ganciclovir
was dissolved in small quantity of distilled
water and the solution was further diluted
with distilled water to mark to obtain a nal
concentration of 100 µg/ml.
Selection of Analytical Wavelengths for ganciclovir
Method A
A 1 ml quantity of 0.5% Sanger’s reagent and
0.2 ml of borate buffer were added to test tube
containing 3.5 ml of ganciclovir and subjected
for heating at 900 C for 10 minutes and cool
the solution, made up to the mark with distilled
water. The absorption spectrums of the complex
were determined against blank solution and the
wavelengths of maximum absorption (λmax) of
the products of the reactions were noted.
Method B
A 1 ml quantity of 0.02% NBS solution, 1.0
ml of 1M HCl were transferred into test tube
and 3.5 ml of ganciclovir stock solution were
added and kept it aside for 20 minutes. Then
add 1 ml of 0.01% methyl orange dye which
results in the formation of pink color complex.
The solutions were made up to 10ml with water.
The absorption spectrum of the complex was
determined against blank solution prepared
without drug. The wavelength of maximum
absorption (λmax) of the product was recorded.
Optimization Studies
Effect of the Sanger’s reagent concentration
In the study of Sanger’s reagent, it revealed
that the reaction was dependent on Sanger’s
reagent concentration. The absorbance of the
reaction solution increased as the Sanger’s
reagent concentration increased, and the
highest absorption intensity was attained at
concentration of 0.5 % (w/v). Higher Sanger’s
reagent concentrations up to 1.0% had no effect

T. Anil Kumar et al., J. Appl. Chem. Res., 20, 1, 14-27 (2012) 17
on the absorption values. Further experiments
were carried out using 0.5 % of the reagent and
results obtained were showed in gure 1.
Figure 1: Effect of Sanger’s reagent (%) on formation of colour product.
Effect of NBS Concentration and hydrochloric
acid
In the study of Sanger’s reagent of NBS reagent,
revealed that the reaction was dependent on
NBS reagent concentration. The absorbance
of the reaction solution was increased as the
NBS concentration increased, and the highest
absorption intensity was attained at NBS
concentration of 0.02 % (w/v). Higher NBS
concentrations up to 0.05 % had no effect on
the absorption values Further experiments were
carried out using 0.02 % and results obtained
were showed in gure 2. It is also observed that
1 ml of 1M hydrochloric acid is necessary for
absorbance and further more addition has no
effect on the absorption (gure 2).
Figure 2: Effect of NBS and HCl on formation of color product

T. Anil Kumar et al., J. Appl. Chem. Res., 20, 1, 14-27 (2012)
18
Procedure for calibration curve
Method A
Aliquates of 0.2 ml, 0.4 ml, --- 0.6 ml of 10 µg/
ml ganciclovir were transferred into different
10 ml volumetric asks, to these solutions 1.0
ml of Sanger’s reagent and 0.2 ml of borate
buffer pH 9 were added. The mixture was
then gently shaken and subjected for heating
at 90o C for 10 minutes and then solution was
cooled. The contents were diluted up to 10ml
with distilled water. The absorbance of each
solution was measured at 353 nm against the
reagent blank prepared in the same manner,
without the analyte. The absorption spectra
and calibration curve are represented in the
gure 3 and 4 respectively.
Figure 3: Absorption spectra of ganci clovir with Sanger’s reagent and NBS reagent .
Figure 4: Linear graph of ganciclovir wit h Sanger’s reagent
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
00.2 0.4 0.6 0.8
Concentration (µg/ml)
Absorbance
Method B
Aliquates of 2 ml, 3 ml, --- 5 ml of 100 µg/ml
ganciclovir were transferred into different 10
ml volumetric asks, to these solutions 1.0 ml
of 0.02% NBS, 1.0 ml of 1M HCl and kept
it aside for 20 minutes and then add 1 ml of
methyl orange indicator. The mixture was then
gently shaken until the appearance of color

T. Anil Kumar et al., J. Appl. Chem. Res., 20, 1, 14-27 (2012) 19
chromogen. The contents were diluted up to
10 ml with distilled water. The absorbance of
each solution was measured at 508 nm against
the reagent blank prepared in the same manner,
without the analyte and the absorption spectra
and calibration curve of ganciclovir is shown
in gure 3 and 5 respectively.
Figure 5: Linear graph of ganciclovir wi th Sanger’s reagent.
0
0.2
0.4
0.6
0.8
1
012345 6
Concentration (µg/ml)
Absorbance
Analysis of commercial pharmaceutical
preparations
Twenty capsules of ganciclovir (Natclovir
and Ganguard) were weighed accurately and
ground into a ne powder. An amount of the
powder equivalent to 100 mg of ganciclovir
was weighed into a 100 ml volumetric ask, 60
ml of water added and shaken thoroughly for
about 20 min. Then, the volume was made up
to the mark with water, mixed well and ltered
through Whatmann lter paper No. 41. First
10 ml portion of the ltrate was rejected and
2.5 ml of the tablet extract was subjected to
analysis using the procedure described above.
Quantication
The limits of the Beer’ law, molar absorptivity
and Sandell’s sensitivity values were evaluated
and are given in Table 1. Regression analyses
of the Beer law plots at their respective λmax
values revealed a good correlation. Graphs of
absorbance versus concentration showed zero
intercept, and are described by the regression
equation, Y = bX + c (where Y is the absorbance
of a 1 cm layer, b is the slope, c is the intercept
and X is the concentration of the drug in μg/
ml) obtained by the least-squares method. The
results are summarized in Table.

T. Anil Kumar et al., J. Appl. Chem. Res., 20, 1, 14-27 (2012)
20
Validation of the method
The validity of the method for the assay of
ganciclovir was examined by determining the
precision and accuracy. This was determined by
analyzing six replicates of the drug within the
Beer’s law limits. The low values of the relative
standard deviation (R.S.D.) indicate good
precision of the methods. To study the accuracy
of the methods, recovery studies were carried out
by the standard calibration curve method. For
this, known quantities of pure ganciclovir were
mixed with denite amounts of pre-analyzed
formulations and the mixtures were analyzed
as before. The total amount of the drug was
then determined and the amount of the added
drug was calculated by difference. The average
present recoveries obtained were quantitative
indicating good accuracy of the methods.
Specicity and selectivity
Ganciclovir solutions were prepared in the
selected media with and without common
exicipients separately. All solutions were
scanned from 800 to 200 nm at a speed of
200 nm min−1 and checked for change in the
absorbance at respective wavelengths. In a
separate study, drug concentration of 0.2 μg/ml
for method A and 2 μg/ml for method B was
prepared independently from pure drug stock
solution in selected media and analyzed paired t
test at 95 % level of signicance was performed
to compare the means of absorbance.
Linearity
To establish linearity of the proposed methods,
a separate series of solutions of ganciclovir
for method A (0.1-0.6 µg/ml) and for method
B (1-5 µg/ml) were prepared from the stock
solutions and analyzed. Least square regression
analysis was performed on the obtained data.
Precision
This good level of precision was suitable for
quality control analysis of the investigated
drug in their pharmaceutical dosage forms.
The precision of the proposed methods was
Table 1. Optical characteri stics of spectrophotometric method.
Parameter Method A Method B
Col or Yellow Pink
λ
max
(nm) 353nm 508 nm
Beer’s law range (μg.ml
-
1
)0.2-0.6 1-5
Molar absorptivity(L.mol
-
1
.cm
-
1
)0.1×10
3
0.32×10
3
Sandell’s Sensitivity (µg.cm
-
2
)0.00068966 0.00624
Limit of detection (µg.ml
-
1
)0.05 0.1045
Limit of quantification (µg.ml
-
1
)0.1619 0.446
Correlation coefficient, R 0.9998 0.9997
Slope b 1.4204 0.789
Intercept a 0.0051 0.0262
Standard deviation of slope 0.03884 0.004203
Standard deviation of intercept 0.002715 0.0007805
Percentage recovery 99.24 99.16

T. Anil Kumar et al., J. Appl. Chem. Res., 20, 1, 14-27 (2012) 21
ascertained by actual determination of six
replicates of xed concentration of the drug
within the Beer’s range and nding out the
absorbance by the proposed method in all the
three drugs. The results are given in Table 2.
Accuracy
To determine the accuracy of the proposed
method, recovery studies were carried out
by adding different known amounts of bulk
samples of ganciclovir within the linearity
range were taken and added to the pre-analyzed
formulation of concentrations 0.2 µg/ml and
2µg/ml for method A and B respectively. The
results are given in Table 3.
Table 2. Accuracy and method precision data for the developed method.
Drug S.No
Label
Claim
(mg)
Amount
found*
%
Purity*
Average
(%) S.D R.S.DaRSDb S.E.M
Method
A
1
250
248.42 99.36
99.30 0.019 0.0716 0.0710 0.013
2247.68 99.10
3250.04 100.02
4249.98 99.90
5250.68 100.27
6248.98 99.59
Method
B
1
250
250.56 100.22
99.56 0.022 0.040 0.0396 0.013
2249.12 99.65
3250.62 100.25
4247.32 98.93
5246.40 98.56
6248.46 99.35
SD. Standard deviation; SEM. Standard error of mean; RSD. Relative standard Deviation;
aintra-day precision, binter-day precision.
Table 3. Standard addition of ganciclovir for accuracy.
formulation
Studied
Amount
taken(µg.ml-1)
Amount
added
Total found
(µg.ml-1)Recovery (%)
Method A 0.2 0.4 0.59 98.46
Method B 2 2 0.38 99.42
Robustness and Ruggedness
To evaluate the robustness of the methods,
reaction time and reagent concentrations were
slightly altered with reference to optimum
values in spectrophotometry. To check the
ruggedness, analysis was performed by four
different analysts; and on three different
spectrophotometers by the same analyst. The
robustness and the ruggedness were checked
at three different drug levels. The intermediate
precision, expressed as percent RSD, which is
a measure of robustness and ruggedness was
within the acceptable limits as shown in the
Table. 2.
Limit of detection (LOD) and limit of

T. Anil Kumar et al., J. Appl. Chem. Res., 20, 1, 14-27 (2012)
22
quantitation (LOQ)
The LOD and LOQ for method A and method B
by the proposed method were determined using
calibration standards. LOD and LOQ were
calculated as 3.3 σ/S and 10 σ/S, respectively,
Where Sis the slope of the calibration curve
and σ is the standard deviation of y-intercept
of regression equation.
Results and Discussion
In method A Sanger’s reagent forms a yellow
color complex with ganciclovir in alkaline
medium and their absorbances were measured
at 353nm. Therefore, the present study
was devoted to explore Sanger’s reagent as
derivatizing reagents for the determination
of ganciclovir in pure and pharmaceutical
dosage forms. The reaction mechanism of
drug Sanger’s reagent shown in the Scheme
1. The method obeys Beer’s law is obeyed
in the range of 0.2-0.6 μg/ml and standard
deviation of slope and intercept were found
to be 0.03884 and 0.002715. Optimization
of the spectrophotometric conditions was
intended to take into account the various goals
of method development. Analytical conditions
were optimized via a number of preliminary
experiments. The effect of Sanger’s reagent
concentration was studied and found that
0.5% gave good absorbance values so further
experiments were carried out using 0.5 %
Sanger’s reagent and effect of heating time on
formation of color product was studied and
different buffer solutions in the pH range of
8.0-10 were tested for reaction of Sanger’s
reagent with ganciclovir. Best results were
obtained in case of borate buffer pH 9.

T. Anil Kumar et al., J. Appl. Chem. Res., 20, 1, 14-27 (2012) 23
Scheme 1: Mechanism of reaction of ganciclovir with Sanger’s.
F
NO
2
NO
2
Sanger's reagent
H
+F+
Yellow colored complex
Intermediate
N
H
N
N
N
H
2
N
OOH
HO
O
Ganciclovir
N
H
N
N
N
NH
OOH
HO
O
F
NO
2
O
2
N
N
H
N
N
N
NH
OOH
HO
O
NO
2
O
2
N

T. Anil Kumar et al., J. Appl. Chem. Res., 20, 1, 14-27 (2012)
24
Method B is based on the oxidation reaction
between ganciclovir and NBS in acidic medium.
The reaction mechanism of drug Sanger’s
reagent shown in the Scheme 2. The Beer’s law
is obeyed in the range of 1-5 μg/ml and standard
deviation of slope and intercept were found
to be 0.004203 and 0.0007805. The method
is indirect and is based on the determination
of residual NBS after having allowed the
oxidation reaction to go to completion under
the specied experimental conditions. The
amount of NBS reacted corresponds to the
drug content in the method. The ability of
NBS to oxidize ganciclovir and bleach the
colors of methyl orange dye has been used for
the indirect spectrophotometric assay of the
drug. In this method, the drug is reacted with
a known excess of NBS in acid medium, and
the unreacted oxidant is determined by reacting
with a xed amount of methyl orange and
measuring the absorbance at 508 nm. In this
method, the absorbance increased linearly with
increasing concentration of drug. Ganciclovir
when added in increasing amounts to a xed
amount of NBS consumes the latter and there
will be a concomitant fall in its concentration.
When a xed amount of dye is added to
decreasing amounts of NBS, a concomitant
increase in the concentration of dye results.
This is observed as a proportional increase in
the absorbance at the respective wavelengths
of maximum absorption with increasing
concentration of ganciclovir as indicated by the
correlation coefcients of 0.9997 respectively.
Scheme 2: Mechanism of reaction of ganciclovir with NBS.
N
H
N
N
N
H2N
O
Ganciclovir
4 NBS Oxidation
N
H
N
N
N
H2N
O
O
Oxidized ganciclovir
4 NBS
N N
H3C
H3C
N S
O
O
O-
N N
H3C
H3C
N S
O
O
O-
Bromination
Br
Br
Br
Br
Methyl Orange
Tetra Bromo Methyl Orange
O
O
O
HO
OH

T. Anil Kumar et al., J. Appl. Chem. Res., 20, 1, 14-27 (2012) 25
Preliminary experiments were performed to
determine the maximum concentrations of the
dye spectrophotometrically, and these were
found to be 0.01%. 1M Hydrochloric acid (1
ml) was the ideal medium for the oxidation
of ganciclovir by NBS as well as the latter’s
determination employing methyl orange dye.
The reaction between ganciclovir and NBS was
unaffected when 1.0 – 2.5 ml of 1 M hydrochloric
acid in a total volume of about 7 ml was used.
Hence, 1.0 ml of 1 M hydrochloric acid is
used for both steps in the assay procedures.
For a quantitative reaction between ganciclovir
and NBS, a contact time of 20 min was found
necessary and constant absorbance readings
were obtained when contact times were extended
upto 20 minutes. A standing time of 5 min was
necessary for the bleaching of the dye color by
the residual NBS. The measured color was found
to be stable for several hours in the presence of
the reaction product (gure 6). Based on various
optical and validation parameters method A is
more sensitive and reliable method compared to
method B.
Figure 6: Effect of reaction time with absorbance.
The results were in agreement with the labeled
amounts. For comparison, a conventional UV
spectrophotometric method developed in our
laboratory was used for parallel comparison.
The recovery percentages for 99.24 and 99.16
for method A and B respectively (Table 4).
This indicated similar accuracy and precision
in the analysis of the investigated compounds
in their pharmaceutical dosage forms.
Table 4. Results of determination of ganciclovir in formulations and statistical comparison with the reference
method.
Pharmaceutical
dosage form
Labelled
Amount
Amount found by
proposed methods
(mg)
Recovery of
Reference
method
Recovery of
proposed methods (%)
Method A
(Natclovir) 250 mg 248.46 98.73
99.38
t=0.566
f=1.084
Method B
(Ganguard) 250 mg 249.12 97.50
99.65
t=0.594
f=1.182

T. Anil Kumar et al., J. Appl. Chem. Res., 20, 1, 14-27 (2012)
26
Conclusion
The reagents utilized in the proposed methods
are cheap, readily available and the procedures
do not involve any critical reaction conditions
or tedious sample preparation. Moreover,
the methods are free from interference by
common additives and excipients. The wide
applicability of the new procedures for routine
quality control was well established by the
assay of ganciclovir in pure form and in
pharmaceutical preparations.
Acknowledgements
The authors express their sincere thanks to
Strides Arco Lab Limited, Bangalore, India
for supplying the gift sample of ganciclovir
(purity-95.3%). Thanks to the Principal, JSS
College of Pharmacy, Mysore, for providing
the necessary facilities.
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