114

IJPAR |Volume 2 | Issue 3 | July-Sep - 2013 ISSN: 2320-2831

Available Online at: www.ijpar.com

[Research article]
Analytical Method Development and Validation for Simultaneous
Estimation of Lamivudine and Zidovudine in Tablet Dosage
form by RP-HPLC
*M.Sravani, K.Haritha Pavani.
Department of Pharmaceutical Analysis, Nimra College of Pharmacy, Nimra Nagar,
Ibrahimpatnam, Vijayawada, A.P, India.

ABSTRACT: A new, precise, rapid, accurate RP-HPLC method was developed for the Simultaneous
Estimation of Lamivudine and Zidovudine in pharmaceutical dosage forms. Chromatographic separation was
achieved with mobile phase consisting of Ammonium acetate buffer pH 4.0, Acetonitrile and THF in the ratio of
60:30:10 v/v as the mobile phase with Inert sil ODS C18 (250 × 4.6 mm I.D) 5 µm, column as stationary phase
at flow rate of 1 mL/min and detection wavelength of 240 nm. The retention times of Lamivudine and
Zidovudine was found to be 3.793 min and 2.547 min respectively. The method was validated in terms of
Linearity, Range, Accuracy, Precision, Specificity, LOD, LOQ, Robustness and system suitability according to
ICH guidelines. Commercial tablet formulation was successfully analyzed using the developed method and the
proposed method is applicable to routine analysis for determination of Lamivudine and Zidovudine in tablet
dosage form.
KEY WORDS: Lamivudine, Zidovudine, RP-HPLC, development, validation, simultaneous estimation.

INTRODUCTION Zidovudine (ZID) is chemically 1-(3-azido-2,3-

dideoxy-β-D-ribofuranosyl)-5-methylpyrimidine-
Lamivudine (LAM) is chemically (2R,5S)-4-
2,4(1H,3H)-dione. Zidovudine is also a nucleoside
amino-1-[2-(hydroxymethyl)-1,3-oxathiolan-5yl]-
analogue which is structurally similar to thymidine
2(1H)-pyrimidinone). Lamivudine is the (-)
and used in the management of AIDS and AIDS-
enantiomer of 2- deoxy-3-thiacytidine, which is a
related complex. It may be given to patients with
nucleoside analog. The (-) enantiomer of the
early HIV infection. In patients with HIV-1
racemic mixture shows much less cytotoxicity than
infected MT-4 cells, Lamivudine in combination
the positive enantiomer. Lamivudine has very low
with zidovudine had synergistic antiretroviral
cellular cytotoxicity and generally less potent than
activity. It is rapidly absorbed from the gastro-
zidovudine in inhibiting HIV-1 and HIV-2
intestinal tract with a bioavailability of about 60–
replication in vitro. It is rapidly absorbed with
70%. It crosses the blood–brain barrier.2
bioavailability of approximately 80%.1

* Corresponding author: M.Sravani.

E-mail address: [email protected]
115
M.Sravani et al / Int. J. of Pharmacy and Analytical Research Vol-2(3) 2013 [114-120]

Fig 1: Chemical structure of LAM Fig 2: Chemical structure of ZID

A literature survey shows that a number of a few HPLC methods for simultaneous estimation
HPTLC4,5,Liquid chromatography tandem mass of LAM and ZID in combined dosage forms were
spectrometry5,6,Uv spectroscopy8 methods have reported9,10,11,12 . But the methods are cost effective.
been reported for the simultaneous estimation of So my aim to develop new method with optimum
LAM and ZID in pharmaceutical dosage forms in pH and less run time for simultaneous estimation of
combination or combinations with other drugs and LAM and ZID in tablet dosage form
Accurately weigh 1.925 gm of Ammonium Acetate
.MATERIALS AND METHODS in 500 mL of HPLC grade water and pH is adjusted
Chemicals and reagents to 4.0±0.2 with O-Phosphoric acid.
Drug samples were obtained from Chandra labs
Preparation of standard stock solution
Pvt. Ltd., India. Tablets (Combivir, Cipla
Stock solution was prepared by dissolving 50 mg
Pharmaceuticals Pvt Ltd), Potassium Dihydrogen
of LAM and 100 mg of ZID in few mL of mobile
orthophosphate (Rankem/AR Grade), Dipotassium
phase in a 100 mLvolumetric flask. It sonicated and
hydrogen orthophosphate (Rankem/AR Grade),
the volume was made to the mark with the mobile
THF (Rankem/AR Grade), ammonium acetate
phase and filtered.
(Rankem/AR Grade), Acetonitrile (Merck/HPLC
Grade), Water (Merck/HPLC Grade), Methanol Working standard solution
(Merck/HPLC Grade), O-phosphoric acid From above stock solution 50 µg/mL of LAM and
(Rankem/ Reagent Grade). 100 µg/mL of ZID working solution was prepared,
pipette out 1 mL in 10 mL volumetric flask and the
INSTRUMENTATION volume up to the mark was made with the mobile
Shimadzu HPLC system equipped with pump (LC- phase. The above solution is used for precision,
10ATVP), UV detector (SPD-10ADVP), column specificity, robustness
Inertsil ODS C18 (250×4.6 mm I.D) 5 µm particle
size, Rheodyne injector fitted with 20µL loop and Sample stock preparation
data read out by using spinchrom software. Twenty tablets were weighed accurately and
powdered. A quantity of powder equivalent to 50
Chromatographic conditions mg of LAM and 100 mg of ZID in 100 mL
Analysis was carried out at ambient temperature. volumetric flask and make up mark with mobile
Compounds were separated isocratically with a phase. The above solution was sonicated and
consisting of ammonium acetate buffer pH 4.0, filtered.
acetonitrile and thf in the ratio of 60:30:10 v/v as
the mobile phase with Inert Sil ODS C18 (250 × Working sample solution
4.6 mm i.d) 5 µm, column as stationary phase at From above stock solution 50 µg/mL of LAM and
flow rate of 1 mL/min and detection wavelength of 100 µg/mL of ZID working solution was prepared
240 nm. The mobile phase was filtered by using by pipette out 1 mL in 10mL volumetric flask and
0.45 µm membrane filter (millipore, bradford, ma), the volume up to the mark was made with the
and sonicated in ultrasonicator for 15 minutes. mobile phase. The above solution is used for assay.
Preparation of Ammonium Acetate buffer Calibration and linearity
pH 4.0±0.2 The calibration curves were constructed in the
range of 30-70 µg/mL for LAM and 60-140 µg/mL
for ZID. the solution were prepared by diluting 0.6,

www.ijpar.com
 116
M.Sravani et al / Int. J. of Pharmacy and Analytical Research Vol-2(3) 2013 [114-120]

0.8, 1.0, 1.2, 1.4 mL of standard stock solution to 0.998 and 0.993, respectively.The linearity graph
10 mL with mobile phase. of LAM and ZID was shown in Fig 1 and Fig 2.

RESULTS AND DISCUSSIONS LOD and LOQ

Optimization of chromatographic conditions by LOD and LOQ is calculated from standard
using different mobile phase compositions. The deviation of response from precision and slope
optimum chromatographic conditions found with from linearity
Ammonium acetate buffer pH 4.0, Acetonitrile and LOQ = 10 σ / S
THF in the ratio of 60:30:10 v/v on Inert sil ODS LOD = 3.3 σ / S
C18 (250 × 4.6 mm I.D) 5 µm particle size with a Where
flow rate of 1 mL/min and detection wavelength σ is standard deviation from response
was selected as 240 nm. LAM was eluted at 3.793 S is slope from calibration curve
min with theoretical plates 3250, tailing factor
The LOD and LOQ for Lamivudine were found to
1.725 and resolution 4.192. ZID was eluted at
be 1.98 µg/mL and 5.99 µg/mL respectively. The
2.547 min with theoretical plates 2320, tailing
LOD and LOQ for zidovudine were found to be
factor 1.742. So this condition was used for assay
3.39 µg/mL and 10.27 µg/mL respectively.
and the assay results were shown in Table 1.
Specificity
Method validation
Specificity of the method was determined by
Method validation done according to the ICH
comparing the retention times of LAM and ZID of
guidelines for validation of analytical procedures
standard solution with the retention times of LAM
System suitability and ZID of sample solutions. Good correlation was
System suitability of method was carried out to obtained between the retention times of standard
verify that the resolution and reproducibility of the with sample shows that there is no interference of
system are satisfactory for the analysis to be excipients from tablet dosage form. Specificity
performed. Theoretical plates, tailing factor, chromatograms given in Fig 3 and Fig 4.
Resolution parameters were determined and
Accuracy
compared against the specifications and are
The degree of accuracy of the method was
presented in Table 2.
determined by recovery studies in triplicate by
Precision standard addition method at 80%, 100% and 120%.
System Precision Known amounts of standard LAM and ZID were
System precision was carried out by using standard added to pre-analyzed samples and were analysed
preparation for six times and the result was in proposed HPLC method. Results of recovery
calculated. The % RSD was found to be less than studies were shown in Table 5 and Table 6.
2%. This proves the method was precise. The result
Robustness
was shown in Table 3.
Robustness of the method was performed by small
Method Precision deliberate variation in operating conditions like
Method precision was carried out by using sample wave length (±2 nm) and flow rate (±0.1mL/min).
preparation for six times and the result was The results were shown in Table 7.
calculated. The % RSD was found to be less than
From linearity the correlation coefficient R2 values
2%, which proves the method was precise. The
were found to be near to 0.999 for both drugs
result was shown in Table 4.
which shows that linear relationship between
Linearity concentrations versus response. The proposed
The linearity of method was studied by preparing HPLC method was also validated for system
different concentration levels of standard solutions. suitability, system precision and method precision.
The linearity range for LAM and ZID were found The % RSD in the peak area of drug was found to
to be 30-70 µg/mL and 60-140 µg/mL, be less than 2%. The number of theoretical plates
respectively. The regression equation for LAM and was found to be more than 2000, which indicates
ZID were found to be y = 26.41x – 57.71 and y = efficient performance of the column. The LOD and
30.80x –1319 with coefficient of correlation, (R2) LOQ for Lamivudine were found to be 1.98 µg/mL
and 5.99 µg/mL respectively. The LOD and LOQ

www.ijpar.com
117
M.Sravani et al / Int. J. of Pharmacy and Analytical Research Vol-2(3) 2013 [114-120]

for zidovudine were found to be 3.39 µg/mL and and ZID were found to be 100.77 and 101.32
10.27 µg/mL respectively, indicates the sensitivity respectively shows that the proposed method is
of the method. The percentage of recovery of LAM highly accurate.

Table: 1 Results for assay

Drug Label claim(mg) Amount found(mg) % Assay

LAM 150 148.09 98.72

ZID 300 298.43 99.48

Table 2 Results for system suitability parameters

Parameters LAM ZID

Retention times(min) 3.793 2.547

Theoretical plates 3250 2320

Tailing factor 1.725 1.742

Resolution 4.192 -
Table 3 Results for system precision

LAM ZID
Injection Retention Retention
Area Area
times times
1 3.833 1165.912 2.583 1467.059
2 3.797 1142.959 2.550 1445.257
3 3.793 1139.959 2.547 1453.436
4 3.780 1149.783 2.537 1448.468
5 3.780 1139.774 2.537 1431.868
6 3.773 1160.209 2.530 1437.808
Average 3.793 1149.766 2.5473 1447.316
SD 0.022 11.064 0.0189 12.344
%RSD 0.57 0.96 0.74 0.85

Table 4 Results for method precision

LAM ZID
Injection Retention Retention
Area Area
times times
1 3.727 1136.462 2.5 1419.877
2 3.767 1134.434 2.537 1421.089
3 3.743 1135.119 2.52 1420.774
4 3.797 1136.717 2.55 1445.257
5 3.78 1143.22 2.537 1437.213
6 3.781 1140.223 2.532 1436.22
Average 3.765833 1137.696 2.529333 1430.072
SD 0.023919 3.073084 0.015808 9.920191
%RSD 0.63517 0.270115 0.624982 0.693685

www.ijpar.com
 118
M.Sravani et al / Int. J. of Pharmacy and Analytical Research Vol-2(3) 2013 [114-120]

Fig 1 Linearity graph of LAM

Fig 2 Linearity graph of ZID

Fig 3 Chromatogram of sample

Fig 4 Chromatogram of standard

www.ijpar.com
119
M.Sravani et al / Int. J. of Pharmacy and Analytical Research Vol-2(3) 2013 [114-120]

Table 5 Results for Recovery of LAM.

Conc Amount Amount added Amount Percentage % Mean

present (µg/mL) found Recovery * Recovery
(µg/mL) (µg/mL)*

50.59
80% 40 10 101.18
100.77

100% 50 10 60.75 101.24

120% 60 10 69.92 99.89

* Mean of three observations

Table 6 Results for Recovery of ZID.

Conc Amount Amount added Amount Percentage % Mean

present (µg/mL) found Recovery * Recovery
(µg/mL) (µg/mL)*
80% 80 20 101.09 101.09
101.32
100% 100 20 121.74 101.45

120% 120 20 141.98 101.42

* Mean of three observations

Table 7 Results for Robustness

Chromatographic changes Retention time(min) Tailing factor

LAM ZID LAM ZID

Flow rate
0.8 4.727 3.157 1.692 1.650
(mL/min)
1 3.797 2.550 1.725 1.710
1.2 3.167 2.113 1.533 1.605
Wavelength
238 3.803 2.540 1.628 1.636
(nm)
240 3.797 2.550 1.725 1.710
242 3.857 2.523 1.600 1.636

* Mean of three observations

CONCLUSION future.
The develop RP-HPLC method was proves to be
precise, rapid, accurate, linear, specific. It can be ACKNOWLEDGEMENT
effectively applied for routine analysis in research The author wish to thank the management of Nimra
institutions, quality control department in College of Pharmacy for providing facilities during
industries, approved testing laboratories in near the work.

www.ijpar.com
 120
M.Sravani et al / Int. J. of Pharmacy and Analytical Research Vol-2(3) 2013 [114-120]

REFERENCES
[1] Lamivudine Drug profile: www.drugbank.ca/drugs/DB00709
[2] Zidovudine drug profile: www.drugbank.ca/drugs/DB00495
[3] D.H. Shewiyo, E. Kaale, C. Ugullum, M.N. Sigond, P.G. Risha, B. Dejaegher, J. Smeyers–Verbeke, Y.
Vander Heyden. Development and validation of a normal-phase HPTLC method for the simultaneous
analysis of lamivudine, stavudine and nevirapine in fixed dosecombination tablets. Journal of
Pharmaceutical and Biomedical Analysis 54 (2011) 445–450.
[4] Palani Venkatesha, Mahesh Daggumati. Development and validation of a 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.
[5] Kathryn B. Kenney, Stephen A. Wring, Richard M. Carr , Guy N. Wells ,John A. Dunn. Simultaneous
determination of zidovudine and lamivudine in human serum using HPLC with tandem mass Spectrometry.
Journal of Pharmaceutical and Biomedical Analysis. 22, 2000, 967–983.
[6] Muralli Krishna Matta, Nageswararao Pilla, Jaswanth Kumar, Laxminarayana.B, Seshagirirao.
Simultaneous quantitation of Lamivudine, Zidovudine and Nevirapine in human plasma by Liquid
chromatography–tandem mass spectrometry and application to a pharmacokinetic study. Acta
Pharmaceutica Sinica B. 2(5), 2012, 472–480.
[7] P. Nagulwar vaishali and P. bhusari kishore . Simultaneous estimation of abacavir, Lamivudine And
Zidovudine in combined tablet dosage form by uv method. Ijrap, 2(2), 2011, 610-614.
[8] Yadavalli Rekha, Yellina Haribabu, Sheeja Velayudhankutty, Sosamma Cicy Eapen and Jane Mary.
Method Development and Validation for the Simultaneous Estimation of Efavirenz, Lamivudine and
Zidovudine through Stability indicating RP-HPLC Method Res. J. Pharmaceutical Sci.2(4), 2013, 10-18.
[9] J. Nijamdeen, B. Jayalakshmi, N. Senthilkumar, Vijayamirtharaj and C. Saravanan. Method development
and validation of RP-HPLC method for simultaneous determination of Lamivudine and Zidovudine. J.
Chem. Pharm. Res., 2010, 2(3), 92-96.
[10] P.Venkatesh, Lydia Smiley and Mahesh D. Simultaneous estimation of Zidovudine and Lamivudine tablets
by RP-HPLC method. International Journal of ChemTech Research 2011, 3(1), 376-380.
[11] Ravi Prakash Mahor, Versha Parcha, Yogendra Singh, Rajiv Sharma and Anil Bhandari. Development and
validation of a HPLC method for simultaneous estimation of Lamivudine and Zidovudine in Tablet Dosage
Forms. Der Chemica Sinica, 2(6), 2011, 12-19.
[12] CH .Venkata Reddiah , P. Rama Devi , K. Mukkanti , K.Srinivasarao. Development and validation of
stability indicating HPLC method for Lamivudine, Zidovudine and Abacavir in tablet dosage forms
Int.J.Pharm.Phytopharmacol.Res 1(5), 2005, 247-256.

*******************************

www.ijpar.com