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Research J. Pharm. and Tech. 1(4): Oct.-Dec.

2008,
,

ISSN 0974-3618 www.rjptonline.org

RESEARCH ARTICLE
Simultaneous RP-HPLC Method for the Estimation of the Emtricitabine, Tenofovir
Disoproxil Fumerate and Efavirenz in Tablet Dosage Forms
N Appala Raju* and Shabana Begum
*
Department of Pharmaceutical Chemistry, Sultan-Ul-Uloom College of Pharmacy, Mount Pleasant, Road-3, Banjara Hills,
Hyderabad-500 034. INDIA

*Corresponding Author E-Mail: rajshaz@gmail.com

ABSTRACT:
A simultaneous stability indicating RP-HPLC method is developed for the estimation of Emtricitabine, Tenofovir disoproxil
fumerate and Efavirenz in tablet dosage form. Chromatography was carried on an Inertsil ODS 3V column using gradient
composition of 0.02M sodium dihydrogen orthophosphaste as mobile phase A and mixture of Methanol and water in ratio of 85:15
as mobile phase B at a flow rate of 1.5 ml/min with detection at 265 nm. The retention times of the Emtricitabine, Tenofovir
disoproxil fumerate and Efavirenz was about 5.875, 8.800 and 12.020 mins respectively. The detector response is linear from 8-
120µg/ml, 12-180µg/ml, 20-360µg/ml of test concentration for Emtricitabine, Tenofovir and Efavirenz respectively. The respective
linear regression equation being Y=10175x-76883 for Emtricitabine, Y=6280.8x+219800 for Tenofovir disoproxil fumerate and
Y=1883.5x+323060 for Efavirenz. The limit of detection and Limit of quantification was 0.06, 0.07 and 0.08 µg/ml and 0.14, 0.12
and 0.15µg/ml for Emtricitabine, Tenofovir and Efavirenz respectively. The percentage assay of Emtricitabine, Tenofovir disoproxil
fumerate and Efavirenz was 99.31, 99.77 and 100.20 % respectively and percentage recovery for average of three different
concentrations was 100.87%, 100.04% and 99.52% respectively. The method was validated by determining its sensitivity, Linearity,
accuracy and precision. The proposed method is simple, fast, sensitive, Linear, accurate, rugged and precise and hence can be
applied for routine quality control of Emtricitabine, Tenofovir disoproxil fumerate and Efavirenz in bulk and in tablet dosage form.

KEY WORDS: Emtricitabine, Tenofovir disoproxil fumerate and Efavirenz, Shimadzu HPLC with PDA detector,
Inertsil ODS 3V and Tablets.

INTRODUCTION:
Emtricitabine2 is chemically known as 4-amino-5-fluoro-1- Literature survey reveals few Chromatographic methods11-13
[(2R, 5S)-2-(hydroxymethyl)-1, 3-oxathiolan-5-yl] in biological fluids were reported along with other
pyrimidin-2-one. Tenofovir disoproxil fumerate fumerate3 is antiretroviral dugs. So far, only one HPLC method has been
chemically know as 9-[(R)-2-[[bis [[isopropoxycarbonyl] reported in gradient mode for the estimation of
oxy] methoxy] phosphonyl] methoxy] popyl] adenine Emtricitabine, Tenofovir disoproxil fumerate and Efavirenz
fumarate. Efavirenz1 is chemically known as (4S)-6-chloro- from pharmaceutical dosage form14.
4-(cyclopropylethynyl)-1, 4-dihydro-4-(trifluoromethyl) 2-
H-3, 1-benzoxazin 2-one. Emtricitabine, Tenofovir Highly Sensitive, Selective, Rugged stability indicating
disoproxil fumerate, and Efavirenz 4,5,6. are a novel HPLC method will be very useful for the estimation of
formulation combining fixed doses of the nucleoside reverse Emtricitabine, Tenofovir disoproxil fumerate and Efavirenz
transcriptase inhibitors Emtricitabine (200mg) and in pharmaceutical formulations. The purpose of the this
Tenofovir disoproxil fumerate fumarate (300mg) with the study was to develop sensitive, simple, precise, accurate and
non-nucleoside reverse transcriptase inhibitor Efavirenz Rugged Simultaneous Stability Indicating RP HPLC method
(600mg) represents the first once-daily, one-tablet for the estimation of Emtricitabine, Tenofovir disoproxil
antiretroviral regimen.10It is official in Martindale2-The fumerate and Efavirenz in bulk drug samples and in
Extra pharmacopoeia. pharmaceutical dosage form.

EXPERIMENTAL:
Materials and Methods:
Received on 05.01.2009 Modified on 10.01.2009 Emtricitabine, Tenofovir disoproxil fumerate and Efavirenz
Accepted on 11.01.2009 © RJPT All right reserved was obtained as a gift sample from Aurobindo Pharma Ltd,
Research J. Pharm. and Tech. 1(4): Oct.-Dec. 2008;Page 522-525 Hyderabad. HPLC grade Methanol, Acetonitrile (Merck)
522
Research J. Pharm. and Tech. 1(4): Oct.-Dec. 2008,
,
and AR grade sodium dihydrogen orthophosphate (Merck) Fig 1:Typical Chromatogram of Emitricetabine (5.88mins),
was used. Milli-Q water was used in mobile phase Tenofovir (8.796) and Efavirenz (12.015) by HPLC
preparation. Commercially available Emtricitabine,
Tenofovir disoproxil fumerate and Efavirenz dosage forms mV

8.796
1750
(Vireday, Cipla) were purchased from local market.
1500

Instrument: 1250
Shimadzu prominence High Performance Liquid
1000
Chromatograph with PDA detector and Auto injector mode

12.015
5.883
750
was used with LC solutions software.
500

Chromatographic conditions: 250


Chromatographic separations were achieved by using 0
Inertsil ODS 3V (250 x 4.6 mm, 5µ) analytical column. The 0.0 2.5 5.0 7.5 10.0 12.5 15.0min
mobile phase is consisting of 0.02M Sodium dihydrogen
orthophosphate monohydrate as mobile phase A and
Methanol and water in the ratio of (85:15) as mobile phase Preparation of Standard Stock solution:
B with gradient program as follows. Accurately 40 mg of Emtricitabine, 60 mg of Tenofovir
disoproxil fumerate and 120 mg of Efavirenz standards were
Gradient Time Program weighed and taken in 25 ml volumetric flask. Dissolved by
Time % A Conc. % B Conc. sonication in 15 ml of Diluent (in a ratio of 70:30
0.01 90 10 Acetonitrile and Buffer) and then diluted to 25 ml with the
Diluent to get 1.6, 2.4 and 4.8mg/ml standard stock solution.
5.00 90 10
6.00 35 65 Working Standard solution:
9.00 10 90 5 ml of the above standard stock solution was taken in 10 ml
11.00 10 90 volumetric flask and made up to 10 ml with diluent to get a
13.00 90 10 concentration of 800, 1200 and 2400 µg/ml for
15.00 90 10 Emtricitabine, Tenofovir disproxil and Efavirenz respectively.
15.01 Stop Stop
Preparation of Sample solution:
Ten tablets (Vireday, Cipla) were accurately weighed and
The flow rate was maintained at 1.5 ml/min with injection
crushed into a fine powder. The powder equivalent to one
volume of 10µl and the absorbance was measured at 265
tablet (200 mg of Emtricitabine, 300 mg of Tenofovir
nm. The column and the HPLC system were kept in ambient
disoproxil fumerate and 600 mg of Efavirenz) was taken in
temperature.
250 ml volumetric flask. About 150 ml diluent was added,
shaken for 5min on rotary shaker and then sonicated for
20mins with intermediate shaking. Then the volume was
Structure of Tenofovir disoproxil, Emtricitabine and Efavirenz. finally made up to the mark (250ml). Sample solution was
centrifuged at 5000 rpm for 5 mins to get a clear solution.
Then supernatant solution was used as final sample solution
of a concentration of 800, 1200 and 2400 µg/ml for the
Emtricitabine, Tenofovir disoproxil fumerate and Efavirenz.

Fig 2 A: Calibration curve of Emtricitabine by HPLC


y = 10175x - 76883
12 2 0 9 78 0

10 3 0 6 0 2 0

Tenofovir Emtricitabine Efavirenz 8402260


Disoproxil.
6 4 9 8 50 0

4 59 4 74 0

Preparation of Mobile phase: 2690980


Sodium Dihydrogen Orthophosphate monohydrate (0.02M)
78 72 2 0
buffer was prepared by dissolving 2.75 gms of buffer in 80 237 394 551 70 8 865 10 2 2 1179
1000 ml of water and by adjusting the pH to 3.5 with dilute C o nc . ( µg/ m L)

orthro phosphoric acid.

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Research J. Pharm. and Tech. 1(4): Oct.-Dec. 2008,
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Table I: Linear Regression Data for Calibration curves:
Drug Emtricitabine Tenofovir Disoprxil Efavirenz
Concentration range (µg/ml) 8 – 120 12 – 180 20 - 360
Slope (m) 10175 6281 1884
Intercept (b) -76883 219800 323060
% Intercept -1.0 2.8 6.7
Residual Sum of Squares 154626 148254 117281
Correlation coefficient 0.9994 0.9994 0.9990
RSQ(r2) 0.999 0.999 0.998
% RSD 0.0 0.1 0.1

Linearity: Fig 2 B: Calibration curve of Tenofovir Disproxil by HPLC


Several aliquots of standard stock solution (0.5, 1.0, 2.5, 3.5,
5.0, 6.0 and 7.5) ml (1 ml=1.6, 2.4 and 4.8 mg/ml of y = 6280.8x + 219800
Emtricitabine, Tenofovir disoproxil fumerate and Efavirenz 1158 3 0 76

respectively) were taken in different 10 ml volumetric flask 9 78 710 0


and diluted up to the mark with Diluent. Evaluation was
79 9 112 4
performed with photo diode detector (PDA) at 265 nm and
Peak area was recorded for all the peaks and a Calibration 6 19 514 8
graph was obtained by plotting peak area versus
4 3 9 9 172
concentration of Emtricitabine (Fig 2 A), Tenofovir
disoproxil fumerate (Fig 2 B) and Efavirenz respectively (Fig 2 6 0 3 19 6
2 C). The plot of peak area of each sample against respective
8 0 72 2 0
concentration was found to be linear in the range of 8 - 120, 12 0 3 15 510 70 5 900 10 9 5 12 9 0 14 8 5 16 8 0
C o n c . ( µ g / mL )
12 - 180 and 20 - 360 µg/ml with correlation coefficient of
0.9994, 0.9994 and 0.9990 and linear regression equation
being Y=10175x-76883, Y=6280.8x+219800 and
Y=1883.5x+323060 for Emtricitabine, Tenofovir disoproxil Table III: Validation Summary: System Suitability as Per USP
fumerate and Efavirenz respectively. Linear regression least
square fit, slope (m), intercept (b), standard deviation, Tenofovir
Emtric
residual sum of squares and correlation coefficient data Drug Disoproxil Efavirenz
itabine
obtained from the measurements are given in Table I. fumerate
Retention Time (mins) 5.875 8.800 12.020
Theoretical Plates (N) 5887 75254 64082
Tailing Factor 0.98 1.23 1.19
Table II: Results of HPLC assay and Recovery studies: Capacity Factor (k') - 0.498 1.046
Tenofovir Resolution - 13.462 20.236
S. Emtric Relative Retention Time 1.00 1.50 2.05
Drug Disoproxil Efavirenz
No. itabine Limit of Detection (µg/ml) 0.06 0.07 0.08
fumerate
Amount claim Limit of Quantification
1 200 300 600 0.14 0.12 0.15
(mg/Tablet) (µg/ml)
2 199.56 300.07 602.05
3 198.64 299.22 600.33 Recovery Studies:
4 Amount Found 198.64 299.90 600.69 Accuracy was determined by adding the known amount of
5 (mg/Tablet) 198.63 299.38 601.22 drug substances of Emtricitabine, Tenofovir disoproxil
6 198.12 298.71 601.50 fumerate and Efavirenz to the pre analyzed samples and
7 198.07 298.57 601.32 subjected to the proposed HPLC analysis. Results of
Mean 198.61 299.31 601.19 recovery study are shown in Table II. The study was done
8 *%Recovery 100.87 100.04 99.51
at three different concentration levels.
* Average of Three different Concentration levels.

RESULTS AND DISCUSSION:


Assay: As per the USP-XXVI system suitability tests were carried
10 µl of sample solution was injected into liquid out on freshly prepared standard stock solution of
chromatograph. The retention time was found to be 5.875 Emtricitabine, Tenofovir disoproxil fumerate and Efavirenz.
mins for Emtricitabine, 8.800 mins for Tenofovir disoproxil Parameters that were studied to evaluate the suitability of
fumerate and 12.020 mins for Efavirenz. The amount of the system are given in Table III. These parameters indicate
drug present per tablet was calculated by comparing the good sensitivity, more ruggedness and robustness of the
respective peak areas of the sample solution with that of the method.
standard solution. The data are presented in Table II.

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Research J. Pharm. and Tech. 1(4): Oct.-Dec. 2008,
,

Limit of Detection (LOD) and Limit of Quantification REFERENCES:


(LOQ): 1. S.Budhavari, The Merck Index (monograph#3521), 14, 598,(2006).
The limit of detection (LOD) and limit of quantification (LOQ) 2. S.Budhavari, The Merck Index (monograph#3565), 14, 606,(2006).
for Emtricitabine, Tenofovir disoproxil fumerate and Efavirenz 3. S.Budhavari, The Merck Index(monograph#9146), 14, 1573(2006)
were found to be 0.06, 0.07 and 0.08 µg/ml and 0.14, 0.12 and 4. C.Sean Sweetman, Martindale-The Complete Drug
0.15µg/ml respectively. The signal to noise ratio is 3 for LOD Reference, 34,632, (2005).
and 10 for LOQ. From the typical chromatogram of 5. C.Sean Sweetman, Martindale-The Complete Drug
Reference, 34,654, (2005).
Emtricitabine, Tenofovir disoproxil fumerate and Efavirenz as 6. Indian pharmacopoeia vol.2, 1071, (2007).
shown in fig 1, it was found that the retention times 5.875 mins 7. Indian pharmacopoeia vol.2, 1075, (2007).
for Emtricitabine, 8.800 mins for Tenofovir disoproxil 8. Indian pharmacopoeia vol.2, 1782, (2007).
fumerate and 12.020 mins for Efavirenz. A gradient program 9. Notari, Stefania; Bocedi, Alessio; Ippolito, Giuseppe;
with mixture of buffer as mobile phase A (0.02M sodium Narciso,Pasquale; Pucillo, Leopoldo Paolo; Tossini, Gianna;
dihydrogen orthro phosphate monohydrate) and methanol and Donnorso, Raffaele Perrone; Gasparrini, Francesco; Ascenzi,
water in a ratio of 85:15v/v as mobile phase B was found to be Paolo, Journal of Chromatography, B: 831(1-2), 258-266,(2006).
most suitable mobile phase combination to obtain well defined 10. Ter Heine, Rob; Alderden-Los, Carolien G.; Rosing, Hilde;
peaks with sharp peak shapes, high theoretical plates and less Hillebrand, Michel J. X.; van Gorp, Eric C. M.; Huitema,
Alwin D. R.; Beijnen, Jos H. Rapid Communications in Mass
tailing. In the present developed HPLC method, the standard Spectrometry, 21(15), 2505-2514 (2007).
and sample preparation involve very simple extraction 11. Rebiere, Herve; Mazel, Bernard; Civade, Corinne; Bonnet, Pierre-
procedure and required very less time. A good linear Antoine, Journal of Chromatography B: 850(1-2), 376-383 (2007).
relationship (more than r=0.999) was observed for 12. Choi, Sun Ok; Rezk, Naser L.; Kashuba, Angela D. M.,
Emitricitabine, Tenofovir Disoproxil fumerate and efavirenz in Journal of Pharmaceutical and Biomedical Analysis, 43(4),
the concentration range of 8 - 120, 12 - 180 and 20 - 360 µg/ml 1562-1567, (2007).
respectively. The percentage assay was found to be 99.31% for 13. Weller, Dennis R.; Brundage, Richard C.; Balfour, Henry H.;
Emtricitabine, 99.77 % for Tenofovir disoproxil fumerate and Vezina, Heather E. Journal of Chromatography, B: 848(2),
100.20% for Efavirenz in tablets. Recovery studies shows good 369-373 (2007).
14. Manogaokar.K, and Desai. A. Indian Drugs 45(3), 188-
extraction and recovery from 50% to 150% of test 192,(2008).
concentration. It was found percentage recovery was about
100.87% for Emtricitabine, 100.04 % for Tenofovir disoproxil
fumerate and 99.51% for Efavirenz indicates good extraction
and good recovery and accuracy of the method. There is no
additional peaks in the chromatogram at the main peak
Retention times indicates non-interference of the common
excipients used in the tablets. This demonstrates that the
developed HPLC method is simple, linear, accurate, sensitive,
rugged and reproducible.

Fig 2 C: Calibration curve of Efavirenz by HPLC


y = 1883.5x + 323060
70 10 73 6

59 4 4 170

4 8 776 0 4

3 8 110 3 8

2 74 4 4 72

16 779 0 6

6 113 4 0
240 605 9 70 13 3 5 170 0 2065 2430 2 79 5 3 16 0 3 52 5
Conc . ( µg/ mL)

Thus, the developed method sensitive, accurate and rugged


and can be easily used for the routine quality control of bulk
and tablet dosage form of Emtricitabine, Tenofovir
disoproxil fumerate and Efavirenz within a short analysis
period of time.

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