Simultaneous Estimation of Tenofovir Disoproxil Fumarate, Efavirenz and Lamivudine in Bulk and Tablet Dosage Form by UV Spectrophotometry

Murugan S.*, Subhashis Debnath, Pranabesh Sikdar, Niranjan Babu M.

Seven Hills College of Pharmacy, Tirupati-517 561, Andhra Pradesh, India

*Corresponding Author E-mail: msm_apcp07@yahoo.com

ABSTRACT:

A simple, sensitive, rapid spectrophotometric method have been developed for simultaneous estimation of Tenofovir disoproxil fumarate (TDF), Efavirenz (EFV) and Lamivudine (LAM) in bulk and in tablet dosage form.The simultaneous equations based on measurement of absorbance at three wavelengths 261 nm, 267 nm and 271 nm λ _max of TDF, EFV and LAM respectively. Beer’s law was obeyed in the concentration range of 2-12 μg/ml, 3-18 μg/ml and 2-12 μg/ml for TDF, EFV and LAM. The methods were validated as per ICH guidelines. Statistical analysis proved that the methods were accurate, precise and reproducible for analysis of LAM, TDF and EFV in tablet dosage form. The wide linearity range, sensitivity, accuracy and simple procedure imply that the proposed technique demonstrated to be appropriate for routine analysis and quality control assay of tablet.

KEYWORDS: Tenofovir disoproxil fumarate, Efavirenz, Lamivudine, Simultaneous equation method

INTRODUCTION:

The multi-drug combinations of nucleoside reverse transcriptase inhibitors (nRTI’s) and non-nucleoside reverse transcriptase inhibitors (NNRTI’s) are effective in the therapy of human immunodeficiency virus (HIV) infection. It is used as a part of highly active anti-retroviral therapy (HAART), for the treatment of HIV^1,2. Three drug FDC comprising of efavirenz, lamivudine, tenofovir disoproxil fumerate form one of the first line regimens in HIV-therapy ³.Efavirenz (EFV), (4S)-6-chloro-4-(cyclopropylethynyl) -4-(trifluromethyl)-1-4-dihydro-2H-3,1-benzoxazin-2-one, is an antiretroviral drug which is a non-nucleoside reverse transcriptase inhibitor (NNRTI)^4,5. EFV has been determined by UV spectroscopic⁶ and RP-HPLC⁷ methods in single and in combined dosage form. Tenofovir disoproxil fumarate (TDF), 9-((R)-2-((bis(((isopropoxycarbonyl)oxy)methoxy)phosphinyl) methoxy)propyl)adenine fumarate (1:1), is a nucleotide analogue reverse transcriptase inhibitor (nRTIs)^4,5. TDF has been determined in spiked human plasma by HPLC^8,9.

The estimation of TDF by RP-HPLC has been reported¹⁰. Lamivudine (LMI), (2R,cis)-4-amino-1-(2-(hydroxylmethyl-1,3-oxathiolan-5-yl)-(1H) pyrimidin-2-one, is nucleoside-reverse transciptase inhibitor (NRTI)^4,5. The estimation of lamivudine using UV ^11,12,13 spectroscopy and HPLC has been reported⁷.

EXPERIMENT:

Instrumentation:

The instrument used in the present study was Shimadzu double beam UV/Visible spectrophotometer (Model UV-1700) with spectral band width of 1 nm. All weighing was done on electronic balance (Model Shimadzu AUX -220).

Reagents and Chemicals:

Analytically pure sample of TDF, EFV and LAM was kindly supplied by Strides Arco Labs. (Bangalore, India). The pharmaceutical dosage form used in this study was a Trioday tablets manufactured by Cipla Ltd (Goa, India) labeled to contain 300 mg of tenofovir disoproxil fumarate, 600 mg of efavirenz and 600 mg of lamivudine I.P.0.1 M NaOH was used as solvent.

Preparation of standard stock solution:

Standard stock solution of lamivudine and tenofovir disoproxil fumarate were prepared by dissolving, 10 mg of lamivudine and tenofovir disoproxil fumarate in 50 ml of 0.1 M NaOH, separately to get a concentration of 200 μg/ml. For standard stock solution of efavirenz, 30 mg was weighed and transferred into 100 ml volumetric flask, dissolved in 0.1 M NaOH and made up to the volume with more 0.1 M NaOH to get a concentration of 300 μg/ml.

Study of spectra and selection of wavelength:

For the selection of analytical wavelength, standard solution of 10 μg/ml TDF, EFV and LAM were prepared separately by appropriate dilution of standard stock solution with 0.1 M NaOH and scanned in the entire UV range to determine λ _max of these drugs.The λ _max of TDF, EFV and LAM were found to be 261 nm, 267 nm and 271 nm, respectively. A series of standard solutions were prepared having concentration range of 2-12 μg/ml for TDF, 3-18 μg/ml for EFV and 2-12 μg/ml for LAM. The absorbance of resulting solutions was measured at 261 nm, 267 nm and 271 nm. The overlain UV absorbance spectrum of TDF, EFV and LAM is shown in Fig. No.1.

Fig.1. Zero order overlain spectra of 10µg/ ml of Tenofovir DF, EFV and LAM respectively

These drugs obeyed linearity in the concentration range under study. Absorptivity values were then determined for these drugs at selected wavelengths. Three simultaneous equations (in three variables c_x, c_y and c_z) were formed using absorptivity coefficient values obtained and are as follows:

Where,

A_1,A₂and A₃ are the absorbance of sample solution at 261 nm, 267 nm and 271 nm respectively.

c_x, c_y and c_z are the concentrations of TDF, EFV and LAM, respectively in mixture.

ax₁, ay₁ and az₁ are absorptivity values of TDF at 261 nm, 267 nm and 272 nm, respectively.

ax₂, ay₂ and az₂ are absorptivity values of EFV at 261 nm, 267 nm and 272 nm, respectively.

ax₃, ay₃ and az₃ are absorptivity values of LAM at 261 nm, 267 nm and 272 nm, respectively.

By applying the cramer's rule to equation 1, 2 and 3 the concentration c_x, c_y and c_z, can be obtained as follows,

Table No 1: Analysis of commercial formulation:

Drug

Sample No.

Labeled amount (mg/tab)

Amount found (mg/tab)*

Percentage obtained*

Average (%)

S.D

% RSD

S.E

LAM

300

302.26

293.59

305.57

300.30

304.63

307.03

100.75

97.86

101.85

100.10

101.54

102.34

100.74

1.6215

1.6096

0.0450

TDF

300

295.24

294.27

295.25

296.80

296.98

293.76

98.41

98.09

98.41

98.93

98.99

97.92

98.46

0.4340

0.4407

0.0120

EFV

600

600.27

607.39

597.29

601.22

597.66

596.63

100.04

101.23

99.54

100.20

99.61

99.43

100.01

0.6679

0.0185

*Mean of Three Observation

Table No 2: Recovery studies:

Drug

Sample No.

Amount present (µg/ml)

Amount added (µg/ml)

Amount estimated* (µg/ml)

Amount recovered (µg/ml)

% Recovery*

S.D

% R.S.D

S.E

LAM

3.0223

2.41

3.00

3.61

5.4400

6.0333

6.6400

2.4177

3.0110

3.6177

100.57

100.22

100.77

0.2775

0.2761

0.0308

Mean

100.52

TDF

2.9538

2.49

2.98

3.53

5.4800

5.8833

6.4233

2.5262

2.9295

3.4695

101.45

98.30

98.28

1.8228

1.8348

0.2025

Mean

99.34

EFV

6.0082

4.79

6.00

7.20

10.7466

12.0333

13.2266

4.7384

6.0251

7.2184

98.73

100.28

100.24

0.8818

0.8840

0.0979

Mean

99.75

*Mean of Three Observation

Analysis of Marketed Tablet Formulation:

For the estimation of drugs in the commercial formulation, twenty tablets were weighed accurately. The average weight was calculated and then crushed to obtain fine powder. A quantity of tablet powder equivalent to about 30 mg of EFV was transferred to 100 ml volumetric flask; 50 ml 0.1 M NaOH was added and sonicated for 15 min, volume was then made up to the mark with 0.1 M NaOH. The resulting solution was mixed and filtered through Whatmann filter paper no 41 and filtrate was appropriately diluted to get approximate concentration of 3 μg/ml of TDF, 6μg/ ml of EFV and 3 μg/ml of LAM, the concentration of TDF, EFV and LAM were determined by measuring absorbance of sample solution at 261 nm, 267 nm and 271 nm and using equations (4), (5) and (6). Concentration of TDF, EFV and LAM in the diluted solution was obtained from calibration curves. Amount of TDF, EFV and LAM in mg/tab was then calculated, by multiplying the concentration obtained with dilution factor. Results of tablet analysis are shown in Table No.1.

Validation:

The proposed methods were validated as per ICH guidelines.

Accuracy:

To ascertain the accuracy of the proposed methods, recovery studies were carried out by standard addition method at three different levels (80%, 100% and 120%). The results of recovery studies, expressed as percent recovery, were satisfactory and are presented in Table No.2

Precision:

The reproducibility of the proposed methods was determined by analyzing tablets at different time intervals on same day in triplicates (intra-day assay precision) and on three different days (inter-day assay precision). Coefficient of variance for intra-day assay precision was found to be 0.1970 (for tenofovir disoproxil fumarate), 0.1665 (for efavirenz) and 0.5014 (for lamivudine) in simultaneous equation method and 0.2136 (for tenofovir disoproxil fumarate), 0.3455 (for efavirenz) and 0.2670 (for lamivudine) in simultaneous equation method.

RESULT AND DISCUSSION:

The method discussed in the present work provide a convenient and reliable way for quantitative determination of TDF, EFV and LAM in combined dose tablet formulation. Wavelength of maximum absorbance for TDF (261 nm), EFV (267 nm) and LAM (272 nm) were selected for analysis by simultaneous equation method. Percent label claim for TDF, EFV and LAM in tablet analysis was found in the range of 98.46 to 100.74 %. Percent recovery for TDF, EFV and LAM, was found in the range of 99.34% to 100.52 % with standard deviation well below 2 indicating accuracy of the methods. Intra-day and Inter-day precision studies were carried out by analyzing tablet formulation, three times on the same day and on three different days,respectively. Standard deviation and coefficient of variance for intra-day and inter-day precision studies was satisfactorily low indicating high degree of precision and reproducibility of this method.

CONCLUSION:

The validated spectrophotometric methods employed here proved to be simple, economical, rapid, precise and accurate. Thus these can be used for routine simultaneous estimation of TDF, EFV and LAM in tablet dosage form instead of processing and analyzing each drug separately.

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Received on 17.12.2012 Modified on 01.01.2013

Research J. Pharm. and Tech. 6(2): Feb. 2013; Page 191-194