Development and Validation of Method for Simultaneous Estimation of Lamivudine, Zidovudine and Nevirapine

 

Jadhav S.D. 1*, Bhatia M.S.2, Thamake S.L.2 and Pishawikar S.A.2

1Department of Pharmaceutical Chemistry, Tatyasaheb Kore College of Pharmacy, Warananagar-416113(M.S.) India.

2Department of Pharmaceutical Chemistry, Bharati Vidyapeeth College of Pharmacy, Morewadi, Kolhapur-416013(M.S.) India.

*Corresponding Author E-mail: swapnil.mpharm@gmail.com

 

ABSTRACT:

Lamivudine (LMV), Zidovudine (ZDV) and Nevirapine (NVR) are used in combination for treatment of HIV I. The present work deals with method development for simultaneous estimation of these drugs in tablet dosage form by UV spectrophotometry. A new simple, accurate and sensitive method for simultaneous estimation of lamivudine, zidovudine and nevirapine from tablet formulation has been developed. The method employs combination of zero order and first order derivative spectroscopy. Estimation of nevirapine was done in zero order derivative mode at 310 nm where zidovudine and lamivudine do not show any interference. Estimation of zidovudine was done in first order derivative mode at 270 nm where lamivudine and nevirapine shows zero crossing points. Estimation of lamivudine is done in zero order derivative mode at 271 nm by correcting the absorbance interferences due to zidovudine and nevirapine.  Absorbance interferences of zidovudine and nevirapine calculated from their absorptivities. The results of analysis validated statistically and recovery studies showed satisfactory results. Thus the method is accurate and reproducible and can be employed for routine analysis of these drugs from tablet dosage forms.

 

KEYWORDS: Lamivudine, Zidovudine, Nevirapine, Absorbance interferences.

 


 

INTRODUCTION:

Lamivudine (LMV) is (2R, cis)-4-amino-1-(2-hydroxymethyl-1,3-oxathiolan-5-yl)-(1H) pyrimidin-2-one. Zidovudine (ZDV) is 3-azido-3’-deoxythymidine, are synthetic nucleoside analogs and Nevirapine (NVR) is 11-cyclopropyl-5, 11-dihydro-4-methyl-6H dipyrido [3,2-b:2’3’-e][1,4]diazepin-6-one, is a non-nucleoside reverse transcriptase inhibitor (NNRTI) of human immunodeficiency virus type 1 (HIV-1).1 Lamivudine is official in IP and USP, Nevirapine is official in IP and Zidovudine is official in IP, BP and EP.2 The combination of these drugs is available in market and used in treatment of HIV-1. Extensive literature survey revealed that various analytical methods reported for simultaneous estimation of lamivudine, zidovudine and nevirapine including HPLC methods and spectrophotometric methods for individual estimation.3-8 But there is no UV spectrophotometry method reported for simultaneous estimation of these drugs in combined dosage forms.

 

MATERIALS AND METHODS:

Instrument:

Spectrophotometric analysis was carried out on a JASCO UV-spectrophotometer 530 using a 1 cm quartz cell. The instrument settings were zero order and first derivative mode and band width of 2.0 nm in the range of 200–350 nm.

 

Reagents and chemicals:

Lamivudine, Zidovudine and Nevirapine supplied by Cipla Ltd. Goa. All solvents were spectrophotometric grade obtained from SD fine chemicals. Water purified by glass distillation apparatus.

 

Method:

Preparation of standard stock solutions:

Standard stock solutions of lamivudine, zidovudine and nevirapine were prepared by dissolving 10 mg pure samples in 100 ml of methanol: water (60:40) to produce concentration 100 µg/ml of lamivudine, zidovudine and nevirapine separately.

 

Selection of sampling wavelength:

Sampling wavelengths were determined by scanning concentrations 10 µg/ml of lamivudine, zidovudine and nevirapine between 200-400 nm. The following spectra analyzed in zero as well as first order derivative mode. From these zero and first order derivative spectras, 310 nm wavelength in zero order derivative mode was selected for estimation of nevirapine where lamivudine and zidovudine not shows any interference (Figure 1)  and 270 nm in first order was selected for estimation of zidovudine at this wavelength lamivudine and zidovudine shows zero crossing points (Figure 2).9 For estimation of lamivudine 271 nm in zero order was selected at this wavelength zidovudine and nevirapine shows considerable absorbance interferences.   These interferences were corrected from absorptivities (A 1%, 1 cm) of zidovudine and nevirapine at 270 nm (Table 1) and their concentrations.

 

FIGURE 1: OVERLAIN SPECTRA OF LAMIVUDINE, ZIDOVUDINE AND NEVIRAPINE IN ZERO ORDER DERIVATIVE MODE.

 

FIGURE 2: OVERLAIN SPECTRA OF LAMIVUDINE, ZIDOVUDINE AND NEVIRAPINE IN FIRST ORDER DERIVATIVE MODE.

 

The standard stock solutions of lamivudine, zidovudine and nevirapine were used to prepare mixed standards. From standard drug solutions six working mixed standard solutions with concentration of 4-24 µg/ml of lamivudine, zidovudine and nevirapine were prepared. The overlain spectrum of working solution was determined. The above six mixed standard solutions were scanned at the selected analytical wavelengths and the calibration curve for lamivudine, zidovudine and nevirapine were constructed. Lamivudine, zidovudine and nevirapine obeyed Beer’s law in the concentration range of 4-24 μg/ml. By using quantitative modes of instrument slope, intercept and correlation coefficient values for calibration curve was obtained for these drugs (Table 2) and the concentration in sample solution was calculated by using formula

                                 

                                Abs. = A + B X C

Where, A= intercept, B= slope and C= concentration.

 

Analysis of commercial formulation:

Tablet analysis was done by taking twenty tablets, triturated and powder equivalent to 15 mg of lamivudine weighed and 5 mg of pure lamivudine was added and dissolved in 60 ml of solvent system sonicated for 10 min, filtered through Whatmann filter paper No. 41 and volume was made up to 100 ml. After appropriate dilutions, the absorbances were measured and the concentration of each was determined with equations generated from calibration curve for respective drugs.

 

Method validation:

The proposed method was validated as per ICH guidelines.10

 

Accuracy and specificity:

Accuracy and specificity of analysis was determined by performing recovery studies by spiking different concentrations of pure drug at 80, 100 and 120 % levels in the preanalyzed tablet sample (Table 3).

 

Precision:

Precision of method was determined by performing repeatability, intraday precision, and interday precision. The results of analysis are given in Table 4.

 

Sensitivity:

Sensitivity of analytical method determined according to ICH guidelines and given by LOD and LOQ.

 

Linearity and range:

The method is linear between concentration 4-24 µg/ml for all drugs. Linearity of method is shown by correlation coefficient.

 

Robustness:

Robustness of this method was determined by using methanol: water (50:50) as a solvent system.

 

RESULTS AND DISCUSSION:

Sampling wavelengths were determined from scanning individual drug samples in 200-350 nm range. Sampling wavelengths were 310 and 271 nm for nevirapine and lamivudine respectively in zero order derivative mode, 270 nm in first order derivative mode for estimation of zidovudine. Results of tablet analysis are reported in Table 3. Method was validated as per ICH guidelines. Results of recovery studies lay between 98- 102% which indicate that method is specific and accurate and the values of standard deviation and RSD (%) for repeatability, intraday and interday were found to be below 2.0 % indicates the method is precise.


TABLE 1: ABSORPTIVITY VALUES FOR ZIDOVUDINE AND NEVIRAPINE.

Concentration (µg/ml)

4

8

12

16

20

24

Mean

Absorptivities 271 nm

ZDV

303.07

304.02

302.88

303.05

303.10

304.0

303.0

NVR

197.71

198.28

197.30

197.52

197.23

198.2

197.7

 

 

TABLE 2: REGRESSION ANALYSIS DATA.

Parameter

LMV

ZDV

NVR

Slope

0.0324

0.0004

0.0064

Intercept

0.0227

0.0010

0.0012

Correlation Coefficient

0.9994

0.9991

0.9992

 

 

TABLE 3: RESULTS OF TABLET ANALYSIS, RECOVERY STUDY AND SENSITIVITY

Analyte

Label claim

(mg/tab)

% Label claim estimated*

(Mean ± S. D.)

% Recovery estimated*

(Mean ± S. D.)

LOD*

(µg/ml)

LOQ*

(µg/ml)

LMV

150

99.90 ± 1.38

100.11 ± 0.83

0.0938

0.2841

ZDV

300

100.24 ± 0.46

100.05 ± 0.27

0.0012

0.0035

NVR

200

100.05 ± 1.15

99.79 ± 0.94

0.0129

0.0391

S.D.: Standard Deviation. * Indicates average of nine determinations.

 

 

TABLE 4: RESULT OF PRECISION STUDIES

Precision

% Label claim estimated* (Mean ± S.D.)

R.S.D. (%)

LMV

ZDV

NVR

LMV

ZDV

NVR

Repeatability

 

100.06±0.77

100.04±0.39

100.98±0.68

0.76

0.39

0.67

Intraday

T-1

100.23±0.78

100.01±0.28

100.72±0.72

0.77

0.29

0.71

T-2

100.34±0.58

99.58±0.94

99.97±0.75

0.57

0.95

0.75

T-3

99.61±0.96

100.21±0.59

98.90±1.22

0.97

0.58

1.23

Interday

Day -1

99.97±1.36

99.82±1.14

100.15±0.87

1.36

1.15

0.86

Day -2

99.97±1.22

100.43±0.67

98.86±1.36

1.22

0.66

1.38

Day -3

99.95±1.37

100.04±1.60

100.50±1.63

1.36

1.60

1.62

S.D.: Standard Deviation, R.S.D.: Relative Standard Deviation, * Indicates average of nine determinations.

 

 


Results of LOD and LOQ reported in Table 3 which indicates that method is sensitive. Linearity of proposed method was found to be linear between concentration 4-24 µg/ml for all three drugs, linearity of method shown by correlation coefficient values near to 0.9999.

 

ACKNOWLEDGEMENT:

Authors are thankful to Cipla Ltd. Goa, for providing the free gift samples of Lamivudine, Zidovudine and Nevirapine. Authors are also thankful to Principal, Tatyasaheb Kore College of Pharmacy, Warananagar and Principal, Bharati Vidyapeeth College of Pharmacy, Kolhapur for providing necessary facilities for this work.

 

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Received on 24.05.2010        Modified on 12.06.2010

Accepted on 24.06.2010        © AJRC All right reserved

Asian J. Research Chem. 3(4): Oct. - Dec. 2010; Page 995-997