Indian Journal of Chemical Technology

Vol. 18, November 2011, pp. 431-438

Spectrophotometric determination of lamuvidine using acidic dye and

coupling reagent
M B Rahul Reddy, B M Gurupadayya * & T Anil Kumar
Department of Pharmaceutical Analysis, JSS College of Pharmacy, JSS University, Mysore 570 015, India
Received 5 October 2010 ; accepted 15 November 2011
Two simple, extraction-free spectrophotometric methods (Method-I and Method-II) for the quantitative estimation of
lamuvidine (LMV) in bulk drug and pharmaceutical formulations (tablets) have been developed. First method is based on the
interaction of LMV with 0.1% methanolic solution of bromophenol blue to form a stable, red-colored, ion-pair complex
showing the peak at 595 nm. Second method is based on the oxidation followed by coupling of 3-methyl-2-benzothiazolinone
hydrazone with LMV in the presence of ferric chloride to form green-colored chromogen exhibiting absorption maximum at
659 nm. Both the methods follow Beers law in the concentration range 1-8 g mL-1. Linear relationships with good correlation
coefficients (0.991-0.9967) are found between absorbance and corresponding concentrations of the drug. The reliability and
performance of the proposed methods are validated statistically, the recovery ranges from 99.4 0.1% to 99.9 0.3%. The
results of analysis for the two methods have been validated statistically and by recovery studies.
Keywords: 3-methyl-2-benzothiazolinone , Acidic dye, Bromophenol blue, Lamivudine, hydrazone, Spectrophotometric study

Lamivudine (LMV), chemically (2R-cis)-4-amino-1(2-hydroxymethyl)-1,3-oxathiolan-5-yl-2-(1H)-pyrithiacytidine, is the active ingredient of many

pharmaceutical preparations concerned with the
treatment of human immune deficiency virus that
causes AIDS1-3. It is also active against hepatitis B
virus. LMV is official in Martindale, the Extra
pharmacopoeia. Literature survey revealed that few
methods are available for the assay of LMV and most
of the methods found in the literature are applicable to
the determination of the drug in body fluid. High
performance liquids chromatography is the most
widely used technique and has been applied for the
determination of LMV in human plasma4-10, rabbit
plasma11, cerebrospinal fluid12, human serum13, blood
cells14 and also for the determination of drug
metabolites in urine15,16. LMV in human serum has
also been determined by capillary electrophoresis17,18.
There are also reports on the development of methods
using chloramines T19, para dimethyl cinnamlehyde20,
para dimethyl benzaldehyde21, potassium permanganate22,
N-bromosuccinamide23, phloroglucinol24, potassium
bromated and bromide mixture25 reagents for the
estimation of LMV in pharmaceuticals. Some of the
reported methods suffer from the use of unstable

*
Corresponding author.
E-mail: bm_guru2004@yahoo.co.in

reagents, expensive chemicals and liquid liquid

extraction22,23(Tables 1 and 2). Some combination
methods developed for drug in combination with
stavudine have been assayed by HPTLC26 and with
abacavir27, zidovudine28 by reverse phase HPLC in
tablet dosage forms. In addition, LMV is also
simultaneously estimated along with stavudine and
nevirapine by reverse phase HPLC method, developed
in tablet dosage forms29. The aim of the present
investigation was to develop a simple and sensitive
visible spectrophotometric method for the quantitation
of LMV in pure drug and in pharmaceutical
formulations. The method uses the well-known
oxidation followed by coupling of 3-methyl-2benzothiazolinone hydrazone (MBTH) with LMV in
the presence of ferric chloride to form green-colored
chromogen exhibiting absorption maximum at 659 nm
and the interaction of LMV with 0.1% methanolic
solution of bromophenol blue (BPB) to form a stable,
red-colored, ion-pair complex peaking at 595 nm. The
structure of lamivudine is represented in Fig. 1.
Experimental Procedure
Apparatus

A UV/VIS spectrophotometer (1800, Pharmaspec,

Shimadzu, Japan) with 1.0 cm matched quartz cells
was used for absorbance measurements.

INDIAN J. CHEM. TECHNOL., NOVEMBER 2011

432

Table 1Comparison of reported HPLC methods with present method

Column

Mobile phase

Linear range, g mL-1

Remarks

Ref.

Phenyl
-

10-5000ng/mL
-

7
8

Octylsilane

Phosphate buffer-CH3CN

0.6-17.6

C-18

Methanol-Water

406.10-4020.05 ng mL-1

Hypersil BDS C-18

Triethyl amine buffer

252000 ng mL-1

BDS Hypersil C-18

MeOH-CH3COONH4

0.5-100

Applicable to human plasma

Applicable to seminal human plasma,
tandem mass spectrometric detection
Applicable to human plasma less
accurate (RE, 0.1-11%)
Applicable to human plasma; involves
protein precipitation prior to separation
Applicable to rabbit plasma and
run time was 15 min
Applicable to metabolite in
human urine; less precise
(RSD 4.7-22.4%)

9
10
11
16

Table 2Comparison of reported spectrophotometric methods with present method

Linear range, g mL-1 Remarks
(E, l mol-1 cm-1)

Reagent

max
nm

Chloramine-T, Methyl orange

Chloramine-T, Indigo carmine
P-Dimethylamino Cinnamaldehyde (PDCA)
P-Dimethylamino Benzaldehyde (PDAB)
Vanillin
KMnO4- fast green FCF

520
610
496
476
474
640

NaIO4-MBTH

620

Iron (III)- Ferricyanide

740

NBS-Celestine Blue

540

Cobalt-thiocyanate

610

Ammonium Molybdate

700

NaNO2-Phloroglucinol

445

0.1-1.0
0.25-3.5
2-10
2-10
2-10
1-8
(1.28 104)
0.6-6.0
(2.02 104)
9.0-75.0
(1.24 103)
0.7-6
(1.6 104)
1.5 - 15
(7.7 103)
11-150
(1.0 103)
2-10

Folin-ciocalteu reagent
Iron (III)-phenanthroline

684
472

2-10
3-15

2.5-7.5 mg

Titrimetric method and less

accurate

BrO-3- Br-1 Methyl Orange

520
610

Method is tedious and time

consuming
Method is tedious and time
consuming

25

BrO-3- Br-1 Indigocarmine

0.125-1.5
(7.4 104)
1.0-8.0
(1.7 104)

Bromophenol Blue

595

1-8
(6.75 103)

No heating/extraction; use no
expensive chemicals; highly
sensitive method

Present
method

MBTH

659

1-8
(2.86 103)

No heating/extraction; use no
expensive chemicals; highly
Sensnsitive method

Present
method

Titration with potassium bromate and Bromide

Ref.

Extraction procedure
Extraction procedure
Less sensitive
Narrow absorption range
Narrow absorption range
Uses an oxidant, which is
unstable in solution
-

19
19
20
21
21
22

Uses an expensive chemical

22

Uses an unstable solution

23

Involves an extraction step

with organic solvent
Requires heating ;
least sensitive
Absorbance measured at shorter
wavelength
Less sensitive
Less sensitive

22

23
23
24
24
24
25

25

REDDY et al.: SPECTROPHOTOMETRIC DETERMINATION OF LAMUVIDINE

433

series of 10 mL calibrated flasks by means of a micro

burette. To all the calibrated flasks, 1 mL of 0.1%
methanolic solution of BPB was added. The solutions
were swirled and allowed to stand for 5 min and the
volume was diluted to the mark with methanol and
mixed well. The absorbance was measured at 595 nm
against the corresponding reagent blank and
calibration graph was constructed.

Fig. 1Chemical structure of lamivudine

Reagents and materials

LMV chemical references (gift sample) used for

this study were obtained from Strides Arcolab
Laboratories Ltd., Bangalore, India; it was reported to
be 99.8% pure and was used as received. All reagents
and solvents used were of analytical grade. The
formulations selected for this study were obtained
from pharmaceutical stores in Mysore and are LMV
brands: Lamivir HBV (Cipla LTD., Sikkim, India),
Hepitec (GSK, India) and Lamidac (Zydus Cadila,
India).
Standard solutions of pure reference LMV
(1000 g mL1) were prepared in methanol for
Method-I and LMV (1000 g mL1) were prepared
in water for Method-II.
A 0.1% bromophenol blue solution (Merck
Specialities Private Limited) was prepared in 95%
pure methanol (Merck Specialities Private Limited),
diluting it in a calibrated flask and filtering using
glass wool. A 1% FeCl3 solution was prepared in
water and filtered using glass wool. A 0.5% MBTH
reagent (Loba Chemie Private Ltd) was prepared in
water and filtered using glass wool.
Standard solutions for Method I and Method II
A stock solution of LMV (1 mg mL1) in methanol
was prepared (MethodI); it was later diluted to
1-8 g mL1 using methanol. A stock solution of
LMV (1 mg mL1) in distilled water was prepared
(Method II); it was later diluted to 1-8 g mL1
using distilled water.
Analytical procedures

Method I (BPB method)

Different aliquots (0.10.8 mL) of the standard
100 g mL1 LMV solution were transferred into a

Method II (MBTH method)

Varying aliquots (0.10.8 mL) of the standard
100 g mL1 LMV solutions were transferred into a
series of 10 mL calibrated flasks by means of a micro
burette. To all the calibrated 10 mL flasks, 1 mL of
0.5% MBTH reagent was added. The solutions were
swirled and allowed to stand for 5 min. A 1 mL of 1%
FeCl3 solution is added to all the flasks. The solutions
were swirled and allowed to stand for 5 min, the
volume was diluted to the mark with distilled water
and finally mixed well. The absorbance was measured
at 659 nm against the corresponding reagent blank
and calibration graph was constructed.
Procedure for tablet

The content of ten tablets (depending on the

content per tablet) was powdered and mixed
thoroughly. An amount of the powder equivalent to
100 mg of active component was weighed into a
100 mL volumetric flask; about 60 mL of methanol
(MethodI), water (MethodII) was added and shaken
thoroughly for about 20 min. The volume was made
up to the mark with respective solvent, shaken and
filtered using filter paper. The filtrate was diluted
sequentially to get 0.1 mg mL1 of the drug.
Results and Discussion
Method development

The methods are based on (Method-I) the

application of acidic dyes for the spectrophotometric
determination of LMV. The structural formula of
LMV feature amine group suggests the use of acidic
dyes (BPB) as chromogenic reagents. The acid dye
technique is a general procedure for the quantitative
analysis of a variety of pharmaceutical amines. In
practice, an aqueous solution containing the amine
and a suitable indicator dye is shaken with an organic
solvent. The concentration of the resulting ion-pair is
then determined spectrophotometrically. Few studies
have reported the analysis of pharmaceutical
compounds through formation of ion-pair, without
extraction,
followed
by
spectrophotometric

434

INDIAN J. CHEM. TECHNOL., NOVEMBER 2011

estimation30. The reaction path way is represented in

Fig. 2. When added in increasing concentrations of
LMV to a fixed concentration of BPB there is a
proportional increase in absorbance at the respective
max. Preliminary experiments were performed to fix
the upper concentrations of the dye that could be
determined spectrophotometrically.
MethodII is based on the oxidation followed by
coupling of 3-methyl-2-benzothiazolinone hydrazone
with LMV in the presence of ferric chloride to form a
green colored chromogen31. Actually, this is an iron
catalyzed oxidative coupling reaction of MBTH with
the drug. Under reaction conditions, on oxidation,
MBTH loses two electrons and one proton forming an
electrophilic intermediate, which is the active
coupling agent32,33. This intermediate undergoes
electrophilic substitution with the drug to form the
colored product. The reaction pathway is represented
in Fig. 3.

Optimization of reaction conditions

Effect of MBTH concentration

The study on MBTH concentrations reveals that
the reaction is dependent on MBTH reagent (Fig. 4).
The absorbance of the reaction solution increases as
the MBTH concentration increases, and the highest
absorption intensity is attained at MBTH
concentration of 0.5% (w/v). A higher MBTH
concentration up to 1.25% has no effect on the
absorption values. Further experiments are carried out
using 0.5% MBTH.
Effect of FeCl3 concentration
The study on FeCl3 concentrations reveals that the
reaction is dependent on FeCl3 reagent (Fig. 4). The
absorbance of the reaction solution increases as the
FeCl3 concentration increases, and the highest
absorption intensity is attained at FeCl3 concentration
of 1% (w/v). A higher FeCl3 concentration up to

Fig. 2Proposal of the reaction pathway between lamuvidine and bromophenol blue

REDDY et al.: SPECTROPHOTOMETRIC DETERMINATION OF LAMUVIDINE

435

Fig. 3Proposal of reaction pathway between lamuvidine and MBTH

Fig. 4 Effect of MBTH and FeCl3 on the reaction with LMV

1.6% has no effect on the absorption values. Further

experiments are carried out using 1% FeCl3.
Effect of BPB concentration
For the first method various concentrations of BPB
are tried and the absorbance maximum is found with
0.1% of BPB. A higher BPB concentration up to 1%
has no effect on the absorption values. Further
experiments are carried out using 0.1% BPB. The
color intensity increases with time. The maximum

Fig. 5 Effect of time on reaction of BPB with LMV

absorbance is seen only after 5 min and this is found

to be stable after 5 min as shown in the Fig. 5.
All the experimental procedures were carried out in
room temperature.
Reaction products

The absorbance spectra of the red colored product

(LMV-BPB) with max of 595 nm and that of the green
colored product (LMV-MBTH) with max 659 nm are
shown in Fig. 6. The above mentioned blanks have
practically negligible absorption in both systems.

INDIAN J. CHEM. TECHNOL., NOVEMBER 2011

436
Analytical data

Method validation
A linear correlation is found between absorbance at
max and LMV concentration. They show negligible
intercept as shown below:
A= 0.050 0.0023 ; R = 0.9913, n=6 (Method I)
A= 0.045 0.0013 ; R = 0.9967, n=7 (Method II)
where A is the absorbance; , the concentration in
g mL-1; R, the regression coefficient; and n, the
number of concentration levels.
Linearity
To establish linearity of the proposed methods, a
separate series of solutions of LMV (1-8 g) has been
prepared from the stock solutions and analyzed. Least
square regression analysis is performed on the
obtained data.
Accuracy
The accuracy of the method is the closeness of the
measured value to the true value for the sample. To
determine the accuracy of the proposed method,

Fig. 6Absorbance spectra of LMV-BPB and LMV-MB reaction

products (initial conc. of LMV 10 g mL-1)

different levels of drug concentrations lower

concentration (LC, 80%), intermediate concentration
(IC, 100%) and higher concentration (HC, 120%)
have been prepared from independent stock solutions
and analyzed. Accuracy is assessed as the percentage
relative error and mean % recovery (Table 3). To
provide an additional support to the accuracy of the
developed assay method, a standard addition method
is employed, which involves the addition of different
concentrations of pure drug to a known pre analyzed
formulation sample and the total concentration is
determined using the proposed methods. The %
recovery of the added pure drug is calculated as %
recovery = [(CtCs)/Ca] 100, where Ct is the
total drug concentration measured after standard
addition; Cs, the drug concentration in the
formulation sample; and Ca, the drug concentration
added to formulation.
Precision
Repeatability is determined by using different
levels of drug concentrations (same concentration
levels taken in accuracy study), prepared from
independent stock solutions and analyzed. Inter-day,
intra-day and inter-instrument variations are studied
to determine intermediate precision of the proposed
analytical methods (Table 4). Different levels of
drug concentrations (6 times) are prepared three
different times in a day and studied for intra-day
variation. The same procedure is followed for three
different days to study inter-day variation. One set of
different levels of the concentrations is re-analyzed
using the Shimadzu 1800 UVVIS spectrophotometer
connected to computer with UV-PC software has been
used to study inter-instrument variation. The
percentage relative standard deviation (% R.S.D.) of
the predicted concentrations from the regression
equation is taken as precision. Precision studies are
also carried out using the real samples of LMV in a
similar way to standard solution to prove the
usefulness of the method.

Table 3Assay of drug in pharmaceutical formulations (tablets)

Formulation

Lamivir
Lamidac
Hepitec
**

Label claim
mg per tablet

150
100
150

Percentage
found** SD

Student
t-value

f-value

BPB

MBTH

Reference method

BPB

MBTH

BPB

MBTH

99.80.55
99.60.45
99.40.24

99.90.54
99.90.43
99.90.22

101.50.92
98.421.18
102.61.54

0.04
0.06
0.02

0.15
0.23
0.44

1.87
1.44
2.33

2.33
1.99
3.01

Mean value of five determinations.

Tabulated t-value at 95% confidence level is 2.77; tabulated f-value at 95% confidence level is 6.39.

REDDY et al.: SPECTROPHOTOMETRIC DETERMINATION OF LAMUVIDINE

437

Table 4Intra-day precision and intra-day error of the methods

LMV, g mL-1

Method

er a, %

RSD, %

CLb, g mL-1

Taken
1

Found
0.9

0.6

0.4

0.9 0.002

BPB

2
3
4

1.92
2.89
3.91

0.13
0.1
0.15

0.2
0.2
0.1

1.98 0.002
2.98 0.004
3.99 0.001

MBTH

5
6
7

4.96
5.88
7.97

0.11
0.25
0.25

0.1
0.1
0.1

4.99 0.003
5.99 0.002
6.99 0.002

er Relative error.
CL Confidence limits at 95% confidence level for seven degrees of freedom.
a
Mean value of seven determinations.
b

Table 5Analytical and validation parameters for the assay of

LMV
Parameter

Method _ I

Method _ II

Color
max, nm
Beers law range, g mL1
Molar absorptivity, L mol-1 cm-1
Limit of detection, g mL1
Limit of quantification, g mL1
Sandells sensitivity, g/cm2
Intercept a Sa
Slope b Sb
(, LMV g mL1)
Correlation coefficient (R)

Red
595
1-8
0.6075 103
0.1
0.3
0.0038
0.050

Green
659
1-8
0.286 103
0.3
0.9
0.0094
0.045

0.0023

0.0013

0.9913

0.9967

Limit of detection (LOD) and limit of quantitation (LOQ)

The LOD and LOQ for Acyclovir, Valacyclovir

and Ganciclovir by the proposed method are
determined using calibration standards. LOD
and LOQ are calculated as 3.3 /S and 10 /S
respectively, where S is the slope of the calibration
curve and is the standard deviation of y-intercept of
regression equation.
The linearity, slope and the intercepts are
calculated using the regression equation. The optical
and validation parameters are listed in Table 5.
Precision and accuracy of the proposed methods are
tested by carrying out the determination of three
replicates of pure and commercial samples of the
drug, whose concentration is within Beers law range.
Values of the standard deviation (SD) and relative
standard deviation (RSD) are calculated. The two
methods have been applied to various pharmaceutical
formulations and recovery studies have been made by
the standard-addition method (Table 3). The results
were in agreement with the labeled amounts. For
comparison, a conventional UV spectrophotometric

method34, is used for parallel comparison. The limit of

detection (LOD) and limit of quantification (LOQ) are
calculated according to the current ICH guidelines35.
Conclusion
Two useful methods for the determination of LMV
have been developed and validated as per ICH
guidelines. The above two methods can be used to
monitor the content uniformity of tablets and purity of
raw material. The proposed methods do not take more
than 15-20 min. The methods could be considered for
the determination of LMV in quality control
laboratories.
Acknowledgement
The authors are thankful to Strides Archo Labs,
Bangalore, India for providing LMV chemical
reference (gift sample) for this study.
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