American Journal of Anal yt ical Chemistry, 2011, 2, 849-856
doi:10.4236/ajac.2011.27097 Published Online November 2011 (http://www.SciRP.org/journal/ajac)
Spectrophotometric Determination of Lamivudine
Using Chloranilic Acid and
2,3-Dichloro-5,6-dicyano-1,4-benzoquinone (DDQ)
Kenneth C. Madu1, P. O. Ukoha1, A. A. Attama2
1Department of Pure and Industrial Chemistry, Faculty of Physical Sciences, University of Nigeria,
Nsukka, Enugu State, Nigeria
2Department of Pharmaceutics, F acul t y of P har m aceutical Sciences , University of Nigeria,
Nsukka, Enugu State, Nigeria
Received June 13, 2011; revised July 20, 2011; accepted August 15, 2011
Abstract
A spectrophotometric method for the assay of lamivudine in pure form and in dosage form was developed in
this study. The method was based on charge-transfer complex formation between the drug, which acted as
n-donor while chloranilic acid and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) acted as a -acceptor
in a non-aqueous solvent in each case. Chloranilic acid was found to form a charge-transfer complex in a 1:1
stoichiometry with lamuvudine (lamivudine-chloranilic acid) with a maximium absorption band at 521 nm.
Also, DDQ was found to form a charge-transfer complex in a 1:1 stoichiometry with lamivudine (lami-
vudine-DDQ) with a maximium absorption band at 530 nm. The pH was obeyed at acid range. The com-
plexes obeyed Beer’s law at a concentration range of 0.04 - 0.28 mg/ml. The thermodynamic parameters
calculated at different temperatures included the molar absorptivity, association constant, free energy change,
enthalpy and entropy. The proposed method has been conveniently applied in the analysis of commercially
available lamivudine tablet with good accuracy and precision.
Keywords: Charge-Transfer Complexation, Chloranilic Acid, DDQ, Lamivudine, Spectrophotometric
Determination, Thermodynamic Studies, pH, Pharmaceutical Formulation
1. Introduction
Lamivudine is chemically 4-amino-1-[(2R, 5S)-2-(hydroxy-
methyl)-1,3-oxathiolan-5-yl] primidin-2-(1H)-one. It is
an antiretroviral drug belonging to the class called nu-
cleoside reverse transcriptase inhibitors (NRTIs)[1]. It
exhibits potent antiretroviral activity [2]. The adult does
is 150 mg three times daily. It was indicated that combi-
nation therapy of lamivudine with zidovudine is associ-
ated with substantial persistent increase in 4
CD

cell
counts and decreases in HIV RNA as measured by po-
lymerase chain reactions [3]. Chloranilic acid, DDQ and
other -acceptors have been variously utilized in the
spectrophotometric assay and analysis of many drugs by
charge-transfer complexation [4-9]. Uv-visible Spectro-
photometric methods are the instrumental methods of
choice which are co mmonly used in industrial labourato-
ries because of their simplicity, accuracy, precision and
low cost [10-12]. In the available literatures, the method
has not been adopted in the analysis of this drug both in
pure sample and in pharmaceutical formulations.
2. Aims and Objectives of the Study
Our contemporar y drug market is frequently eroded with
fake and substandard drugs. Efforts are therefore directed
in this work to the development of simple, accurate and
sensitive analytical methods for screening lamiduvine
which occupy a strategic position in clinical practice.
The aim of the present work is to develop a simple, sen-
sitive and less expensive spectrophotometric method of
analysis for the determination of lamivudine using 2,3-
dichloro-5,6 dicyano-1,4-benzoquinone and chloranilic ac -
id with methanol as the solvent.

K. C. MADU ET AL.
850
3. Experimental
3.1. Materials
The following materials were procured from their local
suppliers and used without further purification: Lami-
vudine pure powder (Fidson Healthcare Ltd, Lagos Ni-
geria), chloranilic acid (Sigma-Aldrich Chemie, Ger-
many), DDQ 98% (Sigma-Aldrich Chemie, Germany),
methanol (Analytical grade, BDH, UK). All other re-
agents and solvents were of analytical grade and were
used as such. All laboratory reagents were freshly pre-
pared.
3.2. Preparation of Standard Solutions
Lamivudine standard solution (0.00372 M): This was
prepared by weighing 0.00853 g accurately on an elec-
tronic weighing balance and dissolving in enough
methanol in 100 ml standard flask and making up to 100
mL with methanol.
Chloranic acid standard solution (0.00372 M): A 0.07
78 g quantity of chloranilic acid was accurately weighed
on an electronic weighing balance and dissolved in
methanol in 100 mL standard flask and the volume made
up to 100 mL with methanol.
2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ 98%)
standard solution (0.00372 M): A 0.0844 g quantity of
DDQ was accurately weighed on an electronic weighing
balance and dissolved in methanol in 100 mL standard
flask and the volu me made up to 100 mL with methanol.
3.3. Absorption Spectra
A 4 mL volume of the chloranilic acid standard solution
was scanned in a double-beam Uv-vis spectrophotometer
from –nm to –nm to determine its wavelength of maxi-
mum absorption. Similarly, the wavelength of maximum
absorption of a coloured solution developed by mixing 4
ml volume of DDQ standard solution and 2 mL of lami-
vudine standard solution was determined, together with a
coloured solution developed on mixing 2 mL of DDQ
standard solution and 2 ml of lamivudine standard solu-
tion.
3.4. Stoichiometric Determination of the
Complex of Lamivudine-Chloranilic
Acid
The slope ratio method was employed. Lamivudine solu-
tion (3.72 × 10–3 M) was kept constant in each case while
chloranilic acid solution (3.72 × 10–3 M) was varied ac-
cording to the following ratios: 0.25:5, 0.25:7.5, ···,
0.25:15 mL of lamivudine:chloranilic acid. They were
transferred into different test tubes from the beaker. The
mixtures were allowed to stand for 1 h before deter-
mining the absorbance at 521 nm against the blank of
methanol and the chloronilic acid. Also, the
chloronilic acid was kept constant while the lami-
vudine was varied as mentioned above. They were
also transferred into different test tubes from the
beaker for colour development and kept for 1 h before
determining the absorbance at 521 nm against the
bl a n k o f m e t h a n ol a n d c h loranilic acid.
3.5. Stoichiometric Determination of the
Complex of Lamivudine-DDQ
The same method described immediately above was
adopted but in this case, the absorbance was determined
at 430 nm.
3.6. Effect of Time on the Formation of
Lamivudine-Chloranilic Acid Complex
The absorbance of a mixture of 2 mL of 3.72 × 10–3 M
lamivudine solution in methanol and 2 mL of 3.72 × 10–3
M chloranilic acid solution in methanol was determined
at various time intervals from 5 s to 120 min at 53l nm
(

max) at room temperature against methanol blank and
the reagent blank.
3.7. Effect of Time on the Formation of
Lamivudine-DDQ Complex
The same method described immediatelyabove was
adopted but in this case, the absorbance was determined
at 430 nm.
3.8. pH Study of Lamivudine-Chloranilic Acid
Complex
A 3.72 × 10–3 M solution of lamivudine was mixed with
a 3.72 × 10–3 M solution of chloranilic acid at the ratio of
2:2 and 6 mL of buffer solution was added in each case
to make up the volume to 10 mL. The same treatment
was done with buffer 1 - 13 in different test tubes for
colour development and kept for 1 h before determining
the absorbance at 521 nm against the blank of methanol,
buffer and the reagent blank.
3.9. pH Study of Lamivudine-DDQ Complex
The same method described immediately above was
adopted but in this case, the absorbance was determined
at 430 nm.

851 K. C. MADU ET AL.
3.10. Determination of Association Constant,
Molar Absorptivity and Thermodynamic
Parameters of the Complexes
Lamivudine–chloranilic acid complex: Serial volumes
(0.4, 0.8, ···, 2.4 mL) of lamivudine solution (3.72 × 10–3
M) were transferred into different test tubes. The solu-
tions were diluted to 3 mL with methanol and serial
volumes (0.4, 0.8, ···, 2.4 mL) of chloranilic acid solution
in methanol (3.72 × 10–3 M) were added to various test
tubes. The content were mixed and left at room tempera-
ture for 1 h after which their absorbance was d etermined
at 521 nm against a blank of methanol and chloranilic
acid at temperature of 30˚C (room temperature). Further
analysis of the reaction mixtures were done by subjecting
them to temperatures of 40˚C, 50˚C and 60˚C in a ther-
mostated water bath.
3.11. For Lamivudine-DDQ
The same method described immediately was adopted
but in this case, the absorbance was determined at 430 nm .
3.12. Beer’s Calibration Plot for
Lamivudine-Chloranilic Acid Complex
Serial concentrations (0.4, 0.8, ···, 2.8 mL) of the stan-
dard solution (3.72 × 10–3 M) were transferred to differ-
ent test tubes. Sufficient volumes of chloranilic acid in
methanol were added to each of the test tubes according
to the stoichiometry determined. Sufficient volumes of
methanol were also added to bring the volumes to 6 mL
in each of the test tubes. The contents were mixed and
left at room temperature for 1 h after which the absorb-
ance of each of the samples was determined at a wave-
length of 521 nm against a methan ol and chloranilic acid
blank. The absorbance values were plotted against the
concentration to obtain the Beer’s calibration curve for
lamivudine.
3.13. For Lamivudine-DDQ Complex
The procedure immediately above was adopted but the
absorbance was determined at 430 nm.
3.14. Assay Procedure for the Complexes
3.14.1. Lamivudine-Chloranilic Acid Complex
One tablet formulation of lamivudine equivalent to 150
mg of lamivudine was crushed in a crucible and dis-
solved in a 50 mL standard flask with methanol to ex-
tract the active pharmaceutical ingredient (API). The
solution was filtered to remove the excipients after shak-
ing for 3 min. The filtrate was made up to 50 mL mark
with methanol. The solution was diluted further by tak-
ing 1 mL of lamivudine and adding 20 mL of methanol.
Serial volumes of 0.4, 1.6 and 2.8 mL of the solution
were transferred into different test tubes. These volumes
give the corresponding concentration 0.06 mg, 0.24 mg
and 0.42 mg respectively as was used in the Beer’s plot
and sufficient amount of methanol added to bring the
volumes to 5 mL each. The constituents were mixed with
sufficient volumes of chloranilic acid and left at room
temperature for 1 h after which their absorbance was
determined at 420 nm against a blank of methanol and
chloranilic acid blank.
3.14.2. Lamivudine-DDQ Complex
The same method described immediately above was
adopted but their absorbance was determined at 430 nm.
4. Results and Discussion
4.1. Absorption Spectra
A solution of chloranilic acid in methanol had a golden
yellow colour with maximum wavelength at 434 nm
(Figure 1). On reacting the colourless drug solution of
lamivudine with chloranilic acid solution, a purple colour
was obtained. This suggested a charge-transfer complex
formation resulting in th e scanning of the complex in the
visible range of 350 - 600 nm, which showed a maxi-
mium peak at 521 nm (Figure 1). The interaction be-
tween lamivudine and chloranilic acid is as shown in
Scheme 1. The complex was formed instantaneously.
The complex was stable for over 24 h as indicated by the
colour of the complex. The plot of absorbance against
time is shown in Figure 2. The absorbance band of
chloranilic acid showed a bathochromic shift (shift to a
longer wavelength).
0.6
0.5
0.4
0.3
0.2
0.1
400 500 600 700
Absorbance
Wavelen
g
th
(
nm
)
Absorption spectra of
lamivudine-chloranilic
acid comple x
Absorption spectra of
chloranilic acid
434
521
Figure 1.

K. C. MADU ET AL.
852
+
H –
+
N – H
Lamivudine Chloranilic acid
Lamivudine-chloranilic acid complex
O
O H
C l
H O
C l
O
Scheme 1. Proposed structe of lamivudine-chloranilic ur
acid charge transfer complex.
Figure 2. Lamivudine-chloranilic acid complex with respect
Also, a solution of DDQ in methanol had a golden
co
.2. Stoichiometric Determination of the
amivudine-chloranilic acid: The stoichiometric ratio of
to absorbance and time.
lour with maximum wavelength at 351 nm (Figure 3).
On reacting the colourless drug solution of lamivudine
with DDQ solution, a deep golden colour was obtained.
This suggested a charge-transfer complex formation re-
sulting in the scanning of the complex in the visible
range of 400 - 600 nm, which showed a maximum peak
at 430 nm (Figure 3). The interaction between lami-
vudine and DDQ is as shown in Scheme 2. The complex
was also formed instantaneously. The complex was sta-
ble for over 24 h as indicated by the colour of the com-
plex. The plot of absorbance against time is shown in
Figure 4. The absorption band of DDQ also showed a
bathochromic shift.
4Complexes
L
0.6 Absorption spectra of DDQ
0.5
0.4
0.3
0.2
0.1
300 400 500 600 700
Absorbance
Wavelength (nm)
Absorption spectra of
lamivu dine-D D
Q
com
p
lex
351 430
Figure 3.
+
Lamivudine
H –
+
N – H
DDQ
Lamivudine-DDQ complex
Scheme 2. Proposed structure of lamivudine-DDQ charge
transfer complex.
Figure 4. Lamivudine-benzoquinone complex with respect
e reactant was determined using slope ratio method. A
to absorbance and time.
th
1:1 ratio of charge-transfer complex was indicated for
the lamivudine-chloranilic acid interaction (Figures 5(a)
and (b)).

K. C. MADU ET AL. 853
dine-DDQ complex: The stoichiometric ratio
ofLamivu the reactant was determined using slope ratio method.
A 1:1 ratio of charge-transfer complex was indicated for
the lamivudine-DDQ interaction (Figures 6(a) and (b)).
(a)
(b)
Figure 5. (a) lamivudine-chnilic acid complex with ex-lora
cess lamivudine; (b) lamivudine-chloranilic acid complex
with excess chloranilic .
(b)
Figure 6. (a) lamivudine-Dlex with excess lami-
amnilic acid complex: They were evalu-
DQ comp
vudine; (b) Lamivudine-DDQ complex with DDQ in excess.
.3. Association Constant, Molar Absorptivity 4and Thermodymamic Parameters of the
Complexes
ivudine-chloraL
ated using modification of the Benesi-Hildebrand Equa-
tion [13] .

  

22 2 2
2
:: :
(: )
11
o
DA DADA
DA o
C
A
D
AEKE
 

 


1

(1)
where [D] and [Ao] are the initial concentrations of the
o
reactants.

2
:
D
A
A

is the absorbance of the complex at
521 nm,

2
:
D
A
E

nm anis the molar absorptivity of the com- plex at 521d

2
D
A
C
K is the stability constant. The
plot of

2)
aga
2(:DA
o
AA

inst


1o D is shown in Fig-
ure 7. Ts and the slopes of the regression
lines were used to obtain the values of

2
:
he intercept
D
A
E

and

2
D
A
C
K respectively, at constant [Ao]. The mosorp-
calculated were almost constant at the different
lar ab
tivities
temperatures. This was expected since ideally, it should
not vary. Increase in temperature may have led to the
dissociation of the formed complexes. These are pre-
sented in Table 1. The standard enthalpy change, Ho, of
the lamivudine-chloranilic acid interaction was obtained
from this equation:

2
:constant
2.303
o
D
LogK A
CH
RT

 (2)
by plotting

2
:
log
D
A
C
K
T, when itagainst the reciprocal of absolute temperature was calculated from the slope of
the regression line. The plot is shown in Figure 8 and the
result is presented in Table 1, where R is the gas con-
stant.
(a)

K. C. MADU ET AL.
854
Figure 8. Logk of Lamivudine-chloranilic acid verses Ther-
modynamic temperature.
Figure 7. Benesi-Hildebrand plots for lamivudine-chloranilic
acid.
Table 1. Logk of Lamivudine-chloranilic acid verses thermodynamic temperature.
K (M–1)
 Log k 1(K–1) Temp (k) G0 cal/deg/mol H0 cal/deg/mol
TS0 cal/deg/mol
6.9.93 0.286 0.1.85 003 303 –10701.315 –67.015 28.92
35.97 0.278 1.56 0.0032 313 –9323.98 –68.93 24.41
2.67 0.250 1.43 0.0031 323 –2637.35 –76.59 6.60
2.55 0.196 1.41 0.0130 333 –2591.65 –97.65 6.018
Sarly, Gibb’s frergy (d the ent
S0) were calculated respectively from the equations 3
an
imile eneG0) anropy
(d 4 and the results are presented in Table 1.
2
(: )
Gln
D
A
C
RT K (3)
oo
GHTS
o
Lamivudine-DDQ complex: The sam
lamivudine-chloranilic acid comp
ab
(4)
e method as in
lex was adopted but the
sorbances were read at 430 nm. The plot of


2
2:
o
A
D
A
A

against 1o D is shown in Figure . The results were
similar to that of lamivudine-chloranilic acid complex
nt
uffer solutions of
H 1 - 13 were used. It was observed that the highest
peak
is
9Figure 9. Benesi-Hildebrand plots for Lamivudine-DDQ.
but differe plots and tables were generated for lami-
vudine-DDQ complex (Figures 9-10 and Table 2).
4.4. pH Studies of the Complexes
Lamivudine-chloranilic acid complex, b
p
peak was at pH of 2.0, which showed that the complex
was favoured under high acidic medium which means
that the complex dissociates appreciably. The plot of
absorbance against pH is as shown in Figure 11.
Lamivudine-DDQ complex: Buffer solutions of pH 1 -
13 were also used. It was observed that the highestFigure 10. Logk of lamivudine-DDQ verses thermodynamic
temperature. at the pH of 6.0 which showed that the complex was

855 K. C. MADU ET AL.
Figure 11. pH study of Lamivudine-chloranilic acid com-
plex.
Figure 12. pH study of lamivudine-DDQ complex.
favoured under a very weak acidic medium which means
that the complex did not dissociate appreciably. The plot
of absorbance against pH is as shown in Figure 12.
4.5. Beer’s Calibration Plots for the Complexes
Lamivudine-chloranilic acid complex: A standard cali-
bration plot for lamivudine was constructed by plotting
absorbance verses concentration of the drug in mg/mL of
the lamivudine standard solution. A straight line passing
through the origin was obtained for the complexed drug,
indicating that spectrophotometric analysis of electron
dono an-
tative analysis of the drug (Figure 13). Conformity
for
mivudine-chloranilic acid complex was adopted but
d
14).
r-acceptor complex formation can be used for qu
ti
with Beer’s law was evident in the concentration rage of
0.04 - 0.28 mg/mL of lamivudine.
Lamivudine-DDQ complex: The same procedure
la
different plot that passed through the origin was obtaine
(Figure
Figure 13. Beer’s calibration curve for lamivudine-chloranilic
acid complex.
Figure 14. Beer’s calibration curve for l amivudine-DDQ com-
plex.
From the assay of the drug, it was discovered that the
recovery experiments carried out on lamivudine in tablet
dosage form showed high quantitative recoveries with
low standard deviations. For lamivudine-chloranilic acid
complex, the mean percentage recovery of the drug was
found to be 85.1%  12.3%, and that of lamivudine-
DDQ, the percentage recovery was 85.1%  12.1% also.
These indicated a high accuracy of the method of analysis.
5. Conclusions
ent reagents (chloranilic acid and DDQ) occurred
i
w
A charge-transfer complexation between lamivudine with
differ
wth a 1:1 stoichiometry in each case, with maximum
avelength of absorption of 521 nm for lamivudine-
chloranilic acid complex and 430 nm for lamivudine-
DDQ complex. The reactions were favoured at acidic

K. C. MADU ET AL.
856
ualitative and quantitative determination of lamivudine
d in dosage form.
medium according to the pH determinations. Thermody-
namically, the complexes were found to be very stable at
room temperature. The methods were used to assay the
drugs in pure form and in dosage form with good preci-
sion and accuracy and can therefore be used in rapid
q
both in pure form an
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