Sindhura and Agarwal et.

al

Indian Journal of Research in Pharmacy and Biotechnology

ISSN: 2321-5674(Print) ISSN: 2320 3471(Online)

ANALYTICAL METHOD DEVELOPMENT AND VALIDATION FOR THE

SIMULTANEOUS ESTIMATION OF LAMIVUDINE, ZIDOVUDINE AND
EFAVIRENZ BY RP-HPLC IN BULK AND PHARMACEUTICAL DOSAGE FORMS
Sindhura D*, Nanda Kishore Agarwal
Department of pharmaceutical analysis, Nimra College of Pharmacy
*Corresponding Author: dasarisindhura.pharma@gmail.com, Phone no: 9951089007
ABSTRACT
A simple rapid, accurate, precise and reproducible validated reverse phase HPLC method was developed for
the determination of Lamivudine, Zidovudine and Efavirenz in bulk and pharmaceutical dosage forms. The
quantification was carried out using Symmetry C18 (250 X 4.6 mm, 5 m) column run in isocratic way using
mobile phase comprising of methanol and water in the ratio of 65:35 v/v and a detection wavelength of 250nm,
and injection volume of 20L, with a flow rate of 1.0mL/min. The retention times of Lamivudine, Zidovudine and
Efavirenz was found to be 2.519, 3.015 and 24.103. The method was validated in terms of linearity, precision,
accuracy, LOD, LOQ and robustness in accordance with ICH guidelines. The linearity ranges of the proposed
method lies between 0.080 mg/mL to 0.120 mg/mL, which is equivalent to 80% to 120% and with correlation
coefficient of r2=0.9995,0.9994 and 0.9993 for Lamivudine, Zidovudine and Efavirenz. The assay of the proposed
method was found to be 99.98%, 99.96% and 100.14%. The recovery studies were also carried out and mean %
Recovery was found to be 100.7%, 100.28%, 100.45%. The % RSD from reproducibility was found to be <2%.
The proposed method was statistically evaluated and can be applied for routine quality control analysis of
Lamivudine, Zidovudine and Efavirenz in bulk and in Pharmaceutical dosage form.
Key Words: Lamivudine, Zidovudine, Efavirenz, RP-HPLC, Symmetry C18, Tablets, Validation.
1. INTRODUCTION
Lamivudine is 4-amino-1-[(2R, 5S)-2(hydroxymethy1)1, 3- oxathiolan-5-yl]-1, 2- dihydro
pyramidine-2-one.The molecular weight is 229.26,
molecular formula is C8H11N3O3S. It is an enantiomer of
dideoxy analogue of cytidine. Zidovudine is 3' azido-3'deoxythymidine. The molecular weight is 267.24,
molecular formula is C10H13N5O4.It is a thymidine
analogue. Both Lamivudine and Zidovudine inhibits the
HIV reverse transcriptase enzyme competitively and acts
as a chain terminator of DNA synthesis and is used in the
treatment of both types of HIV I and HIV II virus and
chronic hepatitis B.Efavirenz is chemically (S)-6-chloro4-(cyclopropylethynyl)-1,4-dihydro4-(trifluoromethyl)2H-3,1- benzoxazin-2-one. The molecular weight is
315.67, molecular formula is C14H9ClF3NO2.It diffuses

Figure.1.Structure of
Lamivudine

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into the cell where it binds adjacent to the active site of

reverse transcriptase. This produces a conformational
change in the enzyme that inhibits function.
Literature review reveals very few methods are
reported for the assay of Lamivudine, Zidovudine and
Efavirenz in Tablet dosage forms using RP-HPLC
method. The reported HPLC methods were having
disadvantages like high flow rate, more organic phase and
use of costly solvents. The proposed RP-HPLC method
utilizes economical solvent system and having advantages
like better retention time, less flow rate, very sharp and
symmetrical peak shapes. The aim of the study was to
develop a simple, precise, economic and accurate RPHPLC method for the estimation of Lamivudine,
Zidovudine and Efavirenz in Tablet dosage forms.

Figure.2. Structure of Zidovudine

Figure.3.Structure of
Efavirenz

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2. MATERIALS AND METHODS

UV-3000 LABINDIA double beam with UV win
5software UV-VISIBLE spectrophotometer with 1cm
matched quartz cells. Schimadzu HPLC equipped with
SPD 20A UV-VIS detector and the column used was
SYMMETRY C18 (250*4.6mm, 5). The data acquisition
was performed by using LC solutions software.
2.1. Chemicals and reagents: Lamivudine, Zidovudine
and Efavirenz pure samples were obtained from Mylan
Laboatories, Hyderabad, India and dosage form DuovirE marketed by CIPLA was purchased from local
pharmacy. Other chemicals all are of HPLC grade.
2.2. Preparation of mobile phase: A suitable quantity of
degassed mixture of methanol and water in the ratio of
65:35 v/v was prepared and filtered through 0.45 filter
under vacuum filtration.
2.3. Preparation of standard solution: Standard
solution of Lamivudine, Zidovudine and Efavirenz
(600g/ml, 150 g/ml,300g/ml) was prepared by
dissolving 60mg of Efavirenz, 15mg of Lamivudine and
30mg of Zidovudine working standard in 50ml of diluent
with sonication and made up to 100ml with the same
diluent.
2.4. Preparation of sample solution: Five tablets were
weighed and finely powdered and a powder quantity
equivalent to 150mg Lamivudine, 300mg of Zidovudine
and 600mg of Efavirenz were accurately weighed and
transferred to a 100ml volumetric flask and 50ml of
diluent was added to the same. The flask was sonicated for
30 min and volume was made up to the mark with diluent.
Transferred 5ml of solution into a 50ml volumetric flask
and dilute up to the mark with diluent so as to obtain a
concentration of 150,300,600 g/mL mixed well and
injected. The amount present in each tablet was calculated
by comparing the area of standard Efavirenz, Lamivudine,
Zidovudine and tablet sample.

2.5. Method optimization: The chromatographic

separation was performed using Symmetry C18
(2504.6mm, 5m) column. For selection of mobile
phase, various mobile phase compositions were
observed for efficient elution and good resolution.
The mobile phase consisting of methanol and water
in the ratio of 65:35v/v was found to be the optimum
composition for efficient elution of analyte. The
mobile phase was injected to the column at a flow
rate of 1.0 ml/min for 35min. The column
temperature was maintained at 30oC. The analyte was
monitored at 250 nm using UV-detector. The
retention time of the drugs was found to be 2.519,
3.015 and 24.103min. Mobile phase was used as
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diluent during the standard and test samples

preparation.
3. RESULTS
3.1. Method Validation
3.1.1. System suitability: System suitability tests are an
integral part of method validation and are used to ensure
adequate performance of the chromatographic system.
Retention time (RT), number of theoretical plates (N) or
column efficiency and tailing factor (T) were evaluated for
five injections of standard solution at a solution of
100g/ml of Lamivudine, Zidovudine and Efavirenz. The
results are tabulated in the table no-2 and the
chromatogram was shown in the figure no- 4.
3.1.2. Specificity: Specificity is the ability of analytical
method to measure accurately and specifically the analyte
in the presence of components that may be expected to be
present in the sample. The specificity of method was
determined by spiking possible impurities at specific level
to standard drug solution (100ppm). The diluent and
placebo solutions were also injected to observe any
interference with the drug peak. The results are tabulated
in the table no-3 and the chromatogram was shown in the
figure no- 5, 6.
3.1.3. Linearity: Linearity is the ability of the method to
produce results that is directly proportional to the
concentration of the analyte in samples with given range.
The linearity of Lamivudine, Zidovudine and Efavirenz
was in the concentration range of 80-120%.From the
linearity studies calibration curve was plotted and
concentrations were subjected to least square regression
analysis to calculate regression equation. The regression
coefficient was found to be 0.9995 for Lamivudine,
0.9994 for Zidovudine and 0.9993 for Efavirenz and
shows good linearity for three drugs. The results are
tabulated in the table no-4 and the chromatogram was
shown in the figure no- 7, 8, 9.
3.1.4. Accuracy: Accuracy is the closeness of results
obtained by a method to the true value. It is the measure of
exactness of the method. Accuracy of the method was
evaluated by standard addition method. Recovery of the
method was determined by spiking an amount of the pure
drug (80%,100% ,120%) at three different concentration
levels in its solution has been added to the pre analyzed
working standard solution of the drug. The results are
tabulated in the table no-5, 6, 7.
3.1.5. Precision: The precision of the analytical method
was studied by analysis of multiple sampling of
homogeneous sample. The Precision expressed as
standard deviation or relative standard deviation.

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3.1.6. System precision: System precision was performed

by injecting a standard solution of Lamivudine,
Zidovudine and Efavirenz at working concentrations five
times. The results are tabulated in the table no-8.
3.1.7. Method precision: Method precision was
performed by analyzing a sample solution of Lamivudine,
Zidovudine and Efavirenz by injecting six replicates of the
same sample preparations at a concentration of
100ppm/mL. The results are tabulated in the table no-9.

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3.1.8. Robustness: Robustness shows the reliability

of an analysis with respect to deliberate variations in
method parameters. If measurements are susceptible
to variations in analytical conditions, the analytical
conditions should be suitably controlled or a
precautionary statement should be included in the
procedure.

Table.1.Optimized chromatogram conditions for Lamivudine, Zidovudine and Efavirenz

Column
Symmetry C18 (250*4.6mm,5)
Mobile phase
Methanol: Water(65:35)
Flow rate
1.0 ml/ min
Wavelength
250 nm
Injection volume
20 l
Column temperature
30o C
Run time
35 min
Table.2. System suitability Data for Lamivudine, Zidovudine and Efavirenz
Lamivudine
Zidovudine
Peak area
Peak area
1
2027423
1501204
2
2025853
1500977
3
2026576
1501113
4
2025030
1501308
5
2027292
1502391
Average
2026435
1501399
SD
1005
568
%RSD
0.05
0.04
Theoretical plates
6615
7512
Tailing factor
1.25
1.32
Retention time
2.519
3.015
S.No

Peak Name
Lamivudine
Zidovudine
Efavirenz

Table.3.Specificity Data for Lamivudine, Zidovudine and Efavirenz

Retention Time (Minutes)
2.541
3.001
22.403

Efavirenz
Peak area
3138895
3142519
3150529
3154259
3158911
3149023
8251
0.26
4500
1.12
24.103

RT ratio
1.00
1.18
8.82

Table.4. Linearity Data for Lamivudine, Zidovudine and Efavirenz

Lamivudine
Zidovudine
Efavirenz
Level
Con. (mg/ml)
Peak area
Con.(mg/ml) Peak area
Con.(mg/ml) Peak area
80%
0.07959
1576950
0.08005
1169205
0.08095
2521629
90%
0.08855
1761010
0.09010
1299358
0.08846
2753981
100%
0.10350
2040551
0.10360
1504208
0.10290
3198155
110%
0.10810
2121575
0.10850
1565537
0.10853
3342493
120%
0.11950
2357552
0.12015
1735081
0.11925
3701006
Slope
19338642.0219
14201079.1883
30526500.1606
Intercept
40602.8714
27753.3632
50253.3069
Correlation coefficient
0.9997
0.9997
0.9997
R square
0.9995
0.9994
0.9993

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S.No
1
2
3
4
5
Average
SD
% RSD

S.No
1
2
3
4
5
6
Average
SD
% RSD

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Table.5. System precision of Lamivudine, Zidovudine and Efavirenz (8)

Lamivudine
Zidovudine
Efavirenz
Area
RT
Area
RT
Area
RT
2027423
2.540
1501204
3.044
3138895
25.192
2025853
2.540
1500977
3.043
3142519
25.100
2026576
2.544
1501113
3.045
3150529
25.061
2025030
2.541
1501308
3.043
3154259
24.954
2027292
2.542
1502391
3.042
3158911
24.559
2026435
1501399
3149023
1005
568
8251
0.05
0.04
0.26
Table.6. Method precision of Lamivudine, Zidovudine and Efavirenz(9)
Lamivudine Assay
RT
Zidovudine
RT
Efavirenz
(%)
Assay (%)
Assay (%)
99.5
2.539
99.3
3.019
99.3
100.5
2.539
99.6
3.019
100.4
99.5
2.541
99.4
3.024
99.3
99.6
2.539
99.8
3.020
99.8
99.8
2.537
99.5
3.020
99.9
100.0
2.540
99.9
3.021
99.9
99.8
99.6
99.8
0.4
0.2
0.4
0.4
0.2
0.4

RT
22.841
22.869
22.940
22.941
22.889
22.941
-

Table.7. Variation in flow rate, column temperature and buffer for Lamivudine (10)
Lamivudine
Flow (mL/min)
Temperature(oC)
Buffer
Low
High
Low
High
Low
High
%RSD
0.05
0.12
0.30
0.03
0.05
0.25
Retention time
3.094
2.556
2.814
2.808
2.809
2.821
Plate count
5.54
6.10
5124
5029
5954
4054
Tailing factor
1.20
1.21
1.20
1.26
1.32
1.22
Parameter

Table.8.Variation in flow rate, column temperature and buffer for Zidovudine (11)
Zidovudine
Flow (mL/min)
Temperature(oC)
Buffer
Low
High
Low
High
Low
High
%RSD
0.14
0.17
0.19
0.06
0.04
0.18
Retention time
3.598
3.000
3.316
3.310
3.291
3.351
Plate count
4818
6010
4523
6842
4898
5818
Tailing factor
1.14
1.21
1.18
1.18
1.27
1.25
Parameter

Table.9.Variation in flow rate, column temperature and buffer for Efavirenz(12)

Efavirenz
Flow (mL/min)
Temperature(oC)
Buffer
Low
High
Low
High
Low
High
%RSD
0.43
0.17
0.23
0.15
0.31
0.23
Retention time
25.251
20.889
23.399
23.677
21.621
26.840
Plate count
4558
5518
5326
5692
5558
4558
Tailing factor
1.32
1.18
1.23
1.32
1.31
1.31
Parameter

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Figure.4.Standard chromatogram

Figure.6.Chromatogram for Specificity

Figure.8.Linearity plot for Zidovudine

4. DISSCUSSION
4.1. System suitability: From the system suitability
studies it was observed that retention time of Lamivudine,
Zidovudine and Efavirenz was found to be 2.519, 3.015
and 24.103 min. % RSD of peak area was found to be
0.05.0.04 and 0.26. Theoretical plates were found to be
more than 4000. USP tailing factor was found to be 1.25,
1.32 and 1.12 for Lamivudine, Zidovudine and Efavirenz.
All the parameters were within the limit.
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Figure.5.Sample chromatogram

Figure.7. Linearity plot for Lamivudine

Figure.9.Linearity plot for Efavirenz

4.2. Specificity: The Chromatograms of Standard and
Sample are identical with nearly same Retention time.
There is no interference with blank and placebo to the
drugs. Hence the proposed method was found to be
specific. 4.3. Linearity: From the Linearity data it was
observed that the method was showing linearity in the
concentration range of 80-120g/ml for Lamivudine,
Zidovudine and Efavirenz. Correlation coefficient was
found to be 0.9995, 0.9994 and 0.9993 for three
compounds.
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4.4. Accuracy: The recoveries of pure drug from the

analyzed solution of formulation were in the range of
99.3%-101.6%, which shows that the method was
accurate.
4.5. Precision
4.5.1. System precision: The percentage relative standard
deviation (RSD) for the peak area of Lamivudine,
Zidovudine and Efavirenz were 0.05, 0.04 and 0.26.
4.5.2. Method precision: The percentage relative
standard deviation for the assay values of Lamivudine,
Zidovudine and Efavirenz were 0.4, 0.2 and 0.4.
4.6. Ruggedness: Comparison of both the results obtained
for two different Analysts shows that the method was
rugged for Analyst-Analyst variability. The %RSD for
intermediate precision for Lamivudine, Zidovudine and
Efavirenz was found to be 0.4, 0.2 and 0.4.
4.7. Robustness

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forms, Journal of Pharmacy Research, 4(10), 2011, 37663768.

Pishawikar SA, Jadhav SD, Bhatia M.s and Thamake SL,
UV spectrophotometric method for the estimation of
Lamivudine, Zidovudine and Nevirapine in pure and tablet
formulation, Asian Journal of Research in Chemistry
Issue, 4(3), 2010, 1-3.
M. Kumar, B. Jayakar, C. Saravanan and M. V.
Kumudhavalli, RP-HPLC method for simultaneous
estimation of Lamivudine and Zidovudine in pure and
tablet formulation, J. Chem. Pharm. Res, 1(2), 2010, 478481.
Palani Venkatesh, M.Ruthu and Y.Padmanabha Reddy,
Normal phase HPTLC method for the simultaneous
analysis of Lamivudine and Zidovudine in fixed dose
combination tablets, Journal of Pharmaceutical Analysis,
2(2), 2012, 152-155.

As the % RSD of retention time and asymmetry

were within limits for variation in flow rate ( 0.1
ml). Hence the allowable flow rate should be
within 0.9 ml to 1.1 ml.
As the % RSD of retention time and asymmetry
were within limits for variation (+ 20C) in column
oven temperature. Hence the allowable variation
in column oven temperature is + 20C.
As the % RSD of retention time and asymmetry
were within limits for variation (+ 2 %) in
composition of mobile phase. Hence the allowable
variation in mobile phase composition is 2 %.
All the system suitability parameters are within
limits for variation (2nm) in wavelength. Hence
the allowable variation in wavelength is 2nm.
The results obtained were satisfactory and are in
good agreement as per the ICH guidelines.

Sigonda, Yogendra Singh and Rajiv Sharma, Normal

phase HPTLC method for the simultaneous analysis of
Lamivudine, Stavudine and Nevirapine in fixed dose
combination tablets, Journal of Pharmaceutical and
Biomedical Analysis, 54(3), 2010, 445-450.

4.8. Acknowledgement: The authors thankful to Mr. K.

Srinivasa Rao (AGM), Mr. Jyothibasu Director, Mylan
Laboratories for providing necessary facilities to carry out
the research work.
5. CONCLUSION

Jacqueline De Souza, Eunice Kazue Kano, Eunice Emiko

Mori Koono, Simone Grigoleto Schramm, Valentina Porta
and Slvia Storpirtis, LCUV Methodology for
Simultaneous Determination of Lamivudine and
Zidovudine in Plasma by LiquidLiquid Extraction,
Chromatographia, 2(69), 2009, 231-235.

Finally it concludes that all the parameters are

within the limits and meet the acceptance criteria of ICH
guidelines for method validation. The proposed method
was simple, accurate, specific, precise, robust, rugged and
economical. Hence this method is validated and can be
used for routine sample analysis
REFERENCES
Balamuralikrishna K, Mahendra K. And B. Syama
Sundar, RP-HPLC method for the simultaneous estimation
of Efavirenz, Lamivudine and Zidovudine in tablet dosage
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www.ijrpb.com

Hemanth Kumar and Anil Bhandari, Reverse-phase high

performance liquid chromatographic method for
simultaneous determination of plasma zidovudine and
nevirapine with UV detection at 260 nm, Journal of
Pharmaceutical and Biomedical Analysis, 29(6), 2002,
1081-1088.
Sindhuri and Versha Parcha, High performance liquid
chromatographic method was developed for the
simultaneous estimation of lamivudine, zidovudine and
nevirapine in pharmaceutical dosage forms, Journal of
Chromatography, 7(2) 2013, 353-362.

Ashok Peepliwal, Sagar D. Vyawahare and Chandrakant

G. Bonde, Quantitative analysis of Zidovudine containing
formulation by FT-IR and UV spectroscopy, Anal.
Methods, 11, 2010, 1756-1763.
Beckett AH, Stenlake JB, Practical pharmaceutical
chemistry. Vol. II. New Delhi: CBS Publisher and
Distributors, 1986, 13-17.
Skoog, DA Holler, FJ Nieman, T.A. Principles of
instrumental analysis, Thomson Brooks/Cole, Fifth
edition. 1998, 329-335.
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