Indo American Journal of Pharmaceutical Research, 2014

ISSN NO: 2231-6876

SIMULTANEOUS ESTIMATION AND VALIDATION OF TENOFOVIR DISPROXIL

FUMARATE,
EMTRICITABINE
AND
RILPIVIRINE
HYDROCHLORIDE
IN
PHARMACEUTICAL DOSAGE FORM BY UV- SPECTROPHOTOMETRY
M. Madhuri, K. Vijaya Sri* and G. Vinay Jain
Department of Pharmaceutical Analysis, Malla Reddy College of Pharmacy, Maisammaguda, Secunderabad-500 014, Andhra
Pradesh, India.

ARTICLE INFO
Article history
Received 14/11/2014
Available online
31/12/2014

Keywords
Uv,
Tenofovir Disproxil Fumarate,
Emtricitabine,
Rilpivirine Hydrochloride,
Simultaneous Equation
Method,
Multi Component Analysis
And Validation.

ABSTRACT
Purpose: The main objective of present research work was to develop and validate the two
UV-spectrophotometric methods for the simultaneous estimation of tenofovir disproxil
fumarate, emtricitabine and rilpivirine hydrochloride in bulk and pharmaceutical dosage
form. Methods: Method A is based on the simultaneous equation and method B on the multicomponent analysis by using methanol: 0.1N HCl (7:3) as a solvent. The simultaneous
equation method depends on mainly that among three components (tenofovir disproxil
fumarate, emtricitabine and rilpivirine hydrochloride), each of which absorbs at the max of
each other. The multi component method mainly based on the total absorbance of a solution
at a given wavelength is equal to the sum of the absorbance of the individual component.
Results: The max of tenofovir disproxil fumarate, emtricitabine and rilpivirine hydrochloride
was found to be 259nm, 291 nm and 280 nm respectively. The linearity range of tenofovir
disproxil fumarate, emtricitabine, and rilpivirine was between 3-21, 1-10 and 0.5-3 g/ml
respectively. The both methods were validated for various parameters as per ICH guidelines
and the results were found to be in acceptable limits. Conclusion: New, simple, accurate
spectrophotometric methods were developed for the simultaneous estimation of tenofovir
disproxil fumarate, emtricitabine and rilpivirine.

Copy right 2014 This is an Open Access article distributed under the terms of the Indo American journal of Pharmaceutical
Research, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Please cite this article in press as M. Madhuri et al. Simultaneous Estimation and Validation of Tenofovir Disproxil Fumarate,
Emtricitabine and Rilpivirine Hydrochloride In Pharmaceutical Dosage Form By Uv- Spectrophotometry. Indo American Journal
of Pharm Research.2014:4(12).

5864

Corresponding author
Dr. K. Vijaya Sri
Malla Reddy College of Pharmacy,
Affliated to Osmania University,
Maisammaguda,
Secunderabad-500 014, TS, India
vijayasree_2002@yahoo.co.in

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INTRODUCTION
Tenofovir disproxil fumarate, emtricitabine and rilpivirine hydrochloride combination is a new formulation consisting of two
nucleoside analog HIV-1 reverse transcriptase inhibitors (emtricitabine, tenofovir disproxil fumarate) and one non nucleoside reverse
transcriptase inhibitor (rilpivirine), is indicated for use alone as a complete regimen for the treatment of HIV-1 infection in
antiretroviral treatment-naive adults with HIV-1 RNA less than or equal to 100,000 copies/ml.[1] Tenofovir disproxil fumarate is
chemically known as 9-[(R)-2-[[bis[[(isopropoxycarbonyl)oxy]methoxy] phosphinyl]- methoxy]propyl]adenine fumarate (1:1).
Tenofovir disproxil fumarate is an acyclic nucleoside phosphonate diester analog of adenosine mono phosphate. Tenofovir disproxil
fumarate requires initial diester hydrolysis for conversion to tenofovir and subsequent phosphorylations by cellular enzymes to form
tenofovir diphosphate. Tenofovir diphosphate inhibits the activity of HIV-1 RT by competing with the natural substrate
deoxyadenosine 5-triphosphate and, after incorporation into DNA, by DNA chain termination. Emtricitabine is chemically known as
5-fluoro-1-[(2R,5S)-2-(hydroxymethyl)-1,3-oxathiolan-5- yl]cytosine. Emtricitabine, a synthetic nucleoside analog of cytidine, is
phosphorylated by cellular enzymes to form emtricitabine 5'-triphosphate. Emtricitabine 5'-triphosphate inhibits the activity of the
HIV-1 RT by competing with the natural substrate deoxycytidine 5'-triphosphate and by being incorporated into nascent viral DNA
which results in chain termination. Rilpivirine hydrochloride is chemically knownas4-[[4-[[4-[(E)-2-cyanoethenyl]-2,6dimethylphenyl]amino]-2pyrimidinyl]amino]benzonitrilemono hydro chloride. Rilpivirine is a diarylpyrimidine non-nucleoside
reverse transcriptase inhibitor of HIV-1. Rilpivirine activity is mediated by non-competitive inhibition of HIV-1 RT.[2] The structures
of tenofovir disproxil fumarate, emtricitabine and rilpivirine hydrochloride are shown in figure 1, 2, 3.

Fig. 1. Structure of Tenofovir disproxil fumarate.

Fig. 2. Structure of Emtricitabine.

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Tenofovir disproxil fumarate and emtricitabine and are official in Indian pharmacopoeia.[3] Rilpivirine hydrochloride is not
official in any pharmacopoeia.
Literature survey reveals that there are several HPLC, UV, LC, LC-MS, HPTLC methods available for estimation of
tenofovir disproxil fumarate, emtricitabine and rilpivirine hydrochloride individually[4-7] and combination[8-12] of tenofovir
disproxil fumarate and emtricitabine. Very few HPLC[13-15] methods available for simultaneous estimation of tenofovir disproxil
fumarate, emtricitabine and rilpivirine hydrochloride. Till date there is no UV spectrophotometric method available for simultaneous
estimation of tenofovir disproxil fumarate, emtricitabine and rilpivirine hydrochloride. Hence there is a need to develop UV
spectrophotometric methods for simultaneous estimation of tenofovir disproxil fumarate, emtricitabine and rilpivirine hydrochloride.

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Fig. 3. Structure of Rilpivirine hydrochloride.

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MATERIALS AND METHODS

Shimadzu Model 1800 UV-Visible double beam spectrophotometer with spectral bandwidth of 0.1nm and a pair of 1cm
matched quartz cells was used in this study. Tenofovir disproxil fumarate, emtricitabine and rilpivirine hydrochloride were kindly
supplied from Hetero Laboratories Ltd. (Hyderabad, A.P, INDIA). Methanol was analytical grade, purchased from Merck Chemical
Company (India). The commercial tablets of tenofovir disproxil fumarate, emtricitabine and rilpivirine hydrochloride combination are
not available in Indian market; hence we have manufactured immediate release tablet containing 300 mg of tenofovir disproxil
fumarate, 200 mg of emtricitabine and 27.5 mg of rilpivirine hydrochloride and acacia, micro crystalline cellouse, lactose, magnesium
stearate and talc.
Preparation of standard stock solution:
Standard stock solution of emtricitabine, tenofovir disproxil fumarate and rilpivirine were prepared separately by dissolving
30 mg of tenofovir disproxil fumarate, 20 mg of emtricitabine in methanol: 0.1N HCl (7:3) and made the volume up to 10 ml with
same solvent and dissolving 25 mg of rilpivirine in methanol: 0.1N HCl (7:3) and made the volume up to 100 ml with same solvent.
(Stock A solution). From the above stock solutions 1ml of the each aliquots were pipetted out in a 10 ml volumetric flask separately
and the volume was made up to the mark with same solvent to obtain the final concentration of 300g/ml of tenofovir disproxil
fumarate, 200g/ml of emtricitabine and 25g/ml of rilpivirine.(stock B solution).
Selection of wavelength:
Working standard solutions of 6g/ml of tenofovir disproxil fumarate, 4g/ml of emtricitabine, and 0.5g/ml of rilpivirine
were further prepared by appropriate dilution of standard stock solutions. Overlain spectra of tenofovir, emtricitabine and rilpivirine
were scanned which is shown in figure 4, from which the wavelengths of 259, 280 and 291 nm were selected for tenofovir, rilpivirine
and emtricitabine respectively for further studies.

Fig. 4. Overlay spectra of tenofovir, emtricitabine, rilpivirine in methanol: 0.1N HCl (7:3).
Method A: Simultaneous Equation Method
Three wavelengths required in this method are selected at which one component shows maximum absorbance, while
remaining shows considerable absorbance. Considering this fact, wavelengths 259nm, 280nm and 291nm (max of all three drugs)
were selected for the estimation of tenofovir disproxil fumarate, emtricitabine and rilpivirine by simultaneous equation method. At all
selected wavelengths the absorbance and absorptivity values of three drugs were determined.[16]
The absorbance of the mixture at 259 nm, 280 nm and 291 nm may be expressed as follows:

For measurements in 1cm cell b = 1. Hence

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Apply the cramers rule for above equations

A1 = ax1Cx + ay1Cy + az1Cz

A2 = ax2Cx + ay2Cy + az2Cz
A3 = ax3Cx + ay3Cy + az3Cz

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A1 = ax1bCx + ay1bCy + az1bCzat 259 nm

A2 = ax2bCx + ay2bCy + az2bCzat 280 nm
A3 = ax3bCx + ay3bCy + az3bCzat 291 nm

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M. Madhuri et al.

D = det

DCx = det

DCy = det

=
=
=
=

ISSN NO: 2231-6876

DCz = det

+
+
+
+

+
=

+
=

+
=
()
+

+
=
()
+
=

Where the absorbance of diluted sample solution at 259, 280 and 291nm are A1, A2and A3 respectively, The absorptivity of
tenofovir at 259, 280 and 291nm are ax1, ax2 and ax3 respectively, The absorptivity of rilpivirine at 259, 280 and 291nm are ay1, ay2
and ay3 respectively, The absorptivity of emtricitabine at 259, 280 and 291nm are az 1, az2 and az3 respectively, Cx, Cy and Cz are the
concentration of tenofovir, rilpivirine and emtricitabine respectively in the diluted samples.
Using above equations 1, 2 and 3 the concentrations of component X( tenofovir), component Y (rilpivirine) and component Z
(emtricitabine) in the sample mixture can be determined.

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Method B: Multicomponent Analysis

In this method the detection of the sample solutions estimated by the Multicomponent Mode of instrument. From the
standard stock solution, seven mixed standards of tenofovir disproxil fumarate, emtricitabine and rilpivirine in the ratio of 1.5:1:0.125,
3:2:0.25, 4.5:3:0.375, 6:4:0.5, 7.5:5:0.675, 9:6:0.75 and 10.5:7:0.875g/ml were prepared by using methanol:0.1 N HCl (7:3) as
solvent. All these mixed standards scanned in the region of 400nm to 200nm in the multi-component mode of instrument as 259, 280,
291nm as sampling wavelengths. The absorbance of mixed standards was processed by means of matrix equations, saved in the
memory of multi component mode of instrument and used as reference for the analysis of sample solution.[17]
The procedure for preparation of sample solution remains same as method A. The sample solution was feed to the instrument
and the concentration of each component was obtained from spectral data of sample solution with reference to the mixed standards.
Validation of developed methods
Linearity
Linearity was evaluated by preparing the solutions having the concentration range of 3-21 g/ml of tenofovir, 1-10 g/ml of
emtricitabine and 0.5-3 g/ml of rilpivirine. The calibration curves were obtained by plotting absorbance against concentration
(g/ml) for three different wavelengths (259nm, 280nm, 291 nm). Standard deviation (SD), slope, intercept, and correlation
coefficient of determinations (r2) of the calibration curves were calculated to ascertain the linearity of the method.

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Application of the Proposed Method for Pharmaceutical Formulation

Twenty tablets were weighed accurately and finely powdered. A powder equivalent to 30mg of tenofovir disproxil fumarate
(20 mg of emtricitabine and 2.5 mg of rilpivirine) was transferred carefully to 100mL volumetric flask. Approximately 75ml of
methanol: 0.1 N HCl (7:3) was added to the flask and mixed well by sonicating it for 10 min. The volume was made up to 100ml with
methanol: 0.1 N HCl (7:3), filtered through what man no. 5 filter paper. The filtrate contains 300 g/ml of tenofovir disproxil
fumarate, 200 g/ml of emtricitabine and 25 g/ml of rilpivirine. 0.1ml of filtrate was taken in a 10ml volumetric flask and made the
volume with methanol: 0.1N HCl (7:3) solvent mixture. The above solution was analyzed at 259, 280 and 291nm wavelengths and
values of the absorbance were substituted in respective equations 1, 2 and 3 to obtain the concentration of tenofovir disproxil
fumarate, rilpivirine and emtricitabine.

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Method precision (repeatability)

The precision of the instrument was checked by repeated scanning and measurement of the absorbance of solution (n=6) for
tenofovir (6 g/ml), emtricitabine (4 g/ml) and rilpivirine (0.5 g/ml) without changing the parameter of the proposed UV method.
The %RSD was calculated.
Intermediate Precision (reproducibility)
The intraday and interday precision of the proposed method was determined by analyzing the corresponding responses on the
same day and next day for three different concentrations of standard solution of tenofovir (6, 12, 18 g/ml), emtricitabine (2, 6,
10g/ml) and rilpivirine (1, 2, 3 g/ml). The result was reported in terms of relative standard deviation (%RSD).
Accuracy
Accuracy of the proposed method was determined using recovery studies by spiking method. The recovery studies were
carried out by adding different amounts (50, 100 and 150%) of the pure drug to the pre-analysed formulation. The solutions were
prepared in triplicates and the % recovery was calculated.
Limit of Detection and Limit of Quantification
The limit of quantification (LOQ) and limit of detection (LOD) were based on the residual standard deviation of the response
and the slope of the constructed calibration curve (n=3), as described in International Conference on Harmonization guidelines Q2
(R1).[18]
LOD = 3.3 /S, LOQ = 10 /S
Where = the standard deviation of the response and S = slope of the calibration curve
Ruggedness Studies
Ruggedness studies were performed by preparing three replicates of 6g/ml of tenofovir, 4g/ml of emtricitabine and
0.5g/ml of rilpivirine and analyzing by two different analysts and on two different instruments and the results are reported as %RSD.
RESULTS & DISCUSSION
The method was validated according to ICH guidelines[19] in order to determine the linearity, precision, accuracy and
ruggedness of the method. The summary of optical parameters are shown in table 1.
Table 1. Summary of optical parameters.
Parameter
Linearity range (g/ml)
Regression equation (y=mx+c)
Slope
Intercept
Correlation coefficient (r)
Molar absorptivity (L mol-1 cm-1)
Precision (%RSD)
Limit of detection
Limit of quantification
Sandells sensitivity
( cm-2/0.001 absorbance units)
90 % Confidence limits
95% Confidence limits

Tenofovir at 259 nm
3-21
y = 0.0468x + 0.014
0.0468
0.014
0.9993
3.1140104
0.294
2.830
8.576
0.020408163

Emtricitabine at 291 nm
1-10
y = 0.1181x + 0.0026
0.1181
0.0026
1
2.9174104
0.479
0.079
0.239
0.008475

Rilpivirine at 280nm
0.5-3
y = 0.3418x-0.0139
0.3418
-0.0139
0.9996
10.5115104
0.878
0.070
0.212
0.003484

0.8827(+), 0.2681(-)
0.9416(+), 0.2093(-)

0.9401(+), 0.2859(-)
1.0027(+), 0.2233(-)

0.9153(+), 0.2534(-) 0.9787

(+),0.1899(-)

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Linearity:
The absorbance of the solutions of tenofovir, emtricitabine and rilpivirine was determined at three wavelengths i.e. 259nm,
280nm, 291nm and the calibration curves were plotted are shown in figure 5, 6 and 7 and the overlay spectras of tenofovir,
emtricitabine and rilpivirine are shown in figure 8, 9 and 10.

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Fig. 5. Calibration curve for Tenofovir disproxil fumarate.

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Fig. 6. Calibration curve for Emtricitabine.

Fig . 7.Calibration curve for Rilpivirine.

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Fig. 8. Overlay spectra of Tenofovir disproxil fumarate.

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Fig. 9. Overlay spectra of Emtricitabine.

Fig. 10. Overlay spectra of Rilpivirine.

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Method precision (repeatability)

Repeatability was determined for all the drugs and % RSD was found to be less than 2 which has shown in table 2.
Table 2. Repeatability.
Drug name
Tenofovir
Emtricitabine
Rilpivirine

Conc
(g/ml
6
4
0.5

1
0.293
0.48
0.160

2
0.294
0.478
0.161

Absorbance
3
4
0.296 0.294
0.477 0.479
0.162 0.161

5
0.295
0.481
0.163

6
0.293
0.479
0.159

Absorbance mean SD

%RSD

0.2940.012
0.4790.0014
0.1610.0014

0.294
0.479
0.878

Intermediate Precision (reproducibility)

The precision of the developed method was expressed in terms of percent relative standard deviation (% RSD). These results
show reproducibility of the assay. The % RSD values were found to be less than 2 that indicate this method precise for the
determination of the pure form. The interday and intraday precision results were mentioned in table 3.
Table 3. Intermediate Precision.
Drug name
Tenofovir

Emtricitabine

Rilpivirine

Concentration
(g/ml)
6
12
18
1
4
8
1
2
3

Intraday precision
Absorbance mean* SD
0.2940.0020
0.5760.0026
0.8510.0031
0.1180.0015
0.4790.0017
0.9450.0025
0.3190.0025
0.6740.0015
0.9150.0030

%RSD
0.680
0.530
0.311
1.298
0.362
0.266
0.829
0.393
0.275

Interday precision
Absorbance mean* SD
0.2940.001
0.5770.002
0.8510.002
0.1180.001
0.4790.001
0.9450.002
0.3190.002
0.6740.001
0.9150.002

%RSD
0.340
0.347
0.235
0.847
0.208
0.212
0.627
0.297
0.219

Accuracy
Accuracy is determined by performing recovery studies at 3 levels in which known amount of analyte shall be added and recovery
shall be carried out in three replicates of each concentration level and the % recovery was calculated. The accuracy results are shown in
table 4.
Table 4. Results of Accuracy.
Drug name

Tenofovir

Emtricitabi
ne
Rilpivirine

Level of
%
Recovery
50
100
150
50
100
150
50
100
150

Amount
taken
(g/ml)
3
3
3
2
2
2
0.25
0.25
0.25

Amount
added
(g/ml)
1.5
3
4.5
1
2
3
0.125
0.25
0.375

Amount found* (g/ml) by

method SD
A
B
4.490.008
4.510.062
6.070.023
6.020.045
7.510.024
7.490.042
2.950.013
3.010.041
3.940.017
4.010.029
4.990.020
4.990.028
0.370.001
0.3760.005
0.500.003
0.500.004
0.630.004
0.620.004

%Recovery* by method SD
A
99.710.173
101.10.388
100.10.323
98.430.448
98.850.426
99.800.400
99.790.287
100.30.577
100.10.606

B
100.261.375
100.350.743
99.900.556
100.261.374
100.360.742
99.910.555
100.271.372
100.350.739
99.900.561

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Ruggedness Studies
Ruggedness was performed by two different analysts and two different instruments and the results of the study were given in
table 5 and % RSD obtained was less than 2 which is within the acceptance limits.

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Limit of Detection and Limit of Quantification

The parameters LOD and LOQ were determined on the basis of response and slope of the regression equation. LOD and
LOQ values are 2.830 and 8.576 for tenofovir, 0.079, 0.239 for emtricitabine and 0.070, 0.212 for rilpivirine respectively.

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Table 5. Ruggedness.
Drug name

Conc
(g/ml)

Tenofovir

6
6
4
4
0.5
0.5

Emtricitabi
ne
Rilpivirine

Parameter
(different
analyst)
Analyst 1
Analyst 2
Analyst 1
Analyst 2
Analyst 1
Analyst 2

Absorbance
mean* SD

%RSD

Parameter
(different instrument)

Absorbance
mean* SD

%RSD

0.2940.0010
0.2950.0010
0.4790.0010
0.4780.0015
0.1610.0006
0.1610.0010

0.340
0.339
0.847
0.319
0.358
0.621

Shimadzu ( UV-1800)
Lab India (UV-3000)
Shimadzu ( UV-1800)
Lab India (UV-3000)
Shimadzu ( UV-1800)
Lab India (UV-3000)

0.2940.0010
0.2940.0020
0.4790.0010
0.4790.0020
0.1610.0006
0.1600.0010

0.340
0.340
0.847
0.418
0.358
0.720

Analysis of Formulation
The % purity of tenofovir, emtricitabine and rilpivirine was 99.45, 100.48 and 101.10 for method A, 99.70, 99.70 and 99.72
for method B respectively and results are shown in table 6.
Table 6. Results of Assay.
Drug name

Labeled claim
(mg)

Tenofovir
Emtricitabine
Rilpivirine

300
200
25

Amount found (mg) by method*

SD
A
B
303.330.022
299.110.012
198.760.004
199.400.008
25.060.003
24.930.0009

%Purity by method* SD
A
101.100.750
99.451.665
100.480.161

B
99.700.383
99.700.382
99.720.394

CONCLUSION
The method A, simultaneous equation method requires measurement of absorbance of all the three drugs at 259 nm, 280 nm
and 291 nm and few simple manual calculations by using simultaneous equations.
The method B, multi component analysis requires three sampling wavelengths (259 nm, 280 nm, 291 nm) and seven mixed standards.
This method has advantages over than method A, as it does not involve any manual calculations. It directly gives the concentration of
the components in the sample mixture by feeding the mixed standards of tenofovir, emtricitabine and rilpivirine in the memory of
Multi component Mode of the instrument
The above proposed UV methods are very simple, precise, accurate, rapid and cost effective for the simultaneous estimation
of tenofovir disproxil fumarate, emtricitabine and rilpivirine from its pharmaceutical dosage forms by the multivariate
spectrophotometric method. Hence it can be utilized for routine analysis in bulk and pharmaceutical dosage forms.

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5872

Recommend future work:

These UV-Spectrophotometric methods applied for ratio spectrophotometry, double divisor spectrophotometry, chemometrics.
ACKNOWLEDGEMENT
The authors are grateful to Chairman, Mallareddy College of Pharmacy for providing necessary research facilities to carry
out the research work and to Hetero drugs, India for providing the gift sample of the drug.

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9. Swapnil AG., Monali SS., Atul HK., Dhaval MP., Vishnu P Ch., Bhanudas SK., Simultaneous determination of emtricitabine and

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