INTRODUCTION:

Emtricitabine² is chemically known as 4-amino-5-fluoro-1-[(2R, 5S)-2-(hydroxymethyl)-1, 3-oxathiolan-5-yl] pyrimidin-2-one. Tenofovir disoproxil fumerate fumerate³ is chemically know as 9-[(R)-2-[[bis [[isopropoxycarbonyl] oxy] methoxy] phosphonyl] methoxy] popyl] adenine fumarate. Efavirenz¹ is chemically known as (4S)-6-chloro-4-(cyclopropylethynyl)-1, 4-dihydro-4-(trifluoromethyl) 2-H-3, 1-benzoxazin 2-one. Emtricitabine, Tenofovir disoproxil fumerate, and Efavirenz ^4,5,6. are a novel formulation combining fixed doses of the nucleoside reverse transcriptase inhibitors Emtricitabine (200mg) and Tenofovir disoproxil fumerate fumarate (300mg) with the non-nucleoside reverse transcriptase inhibitor Efavirenz (600mg) represents the first once-daily, one-tablet antiretroviral regimen.¹⁰It is official in Martindale²-The Extra pharmacopoeia.

Literature survey reveals few Chromatographic methods^11-13 in biological fluids were reported along with other antiretroviral dugs. So far, only one HPLC method has been reported in gradient mode for the estimation of Emtricitabine, Tenofovir disoproxil fumerate and Efavirenz from pharmaceutical dosage form¹⁴.

Highly Sensitive, Selective, Rugged stability indicating HPLC method will be very useful for the estimation of Emtricitabine, Tenofovir disoproxil fumerate and Efavirenz in pharmaceutical formulations. The purpose of the this study was to develop sensitive, simple, precise, accurate and Rugged Simultaneous Stability Indicating RP HPLC method for the estimation of Emtricitabine, Tenofovir disoproxil fumerate and Efavirenz in bulk drug samples and in pharmaceutical dosage form.

EXPERIMENTAL:

Materials and Methods:

Emtricitabine, Tenofovir disoproxil fumerate and Efavirenz was obtained as a gift sample from Aurobindo Pharma Ltd, Hyderabad. HPLC grade Methanol, Acetonitrile (Merck) and AR grade sodium dihydrogen orthophosphate (Merck) was used. Milli-Q water was used in mobile phase preparation. Commercially available Emtricitabine, Tenofovir disoproxil fumerate and Efavirenz dosage forms (Vireday, Cipla) were purchased from local market.

Instrument:

Shimadzu prominence High Performance Liquid Chromatograph with PDA detector and Auto injector mode was used with LC solutions software.

Chromatographic conditions:

Chromatographic separations were achieved by using Inertsil ODS 3V (250 x 4.6 mm, 5µ) analytical column. The 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 B with gradient program as follows.

Gradient Time Program
Time	% A Conc.	% B Conc.
0.01	90	10
5.00	90	10
6.00	35	65
9.00	10	90
11.00	10	90
13.00	90	10
15.00	90	10
15.01	Stop	Stop

The flow rate was maintained at 1.5 ml/min with injection volume of 10µl and the absorbance was measured at 265 nm. The column and the HPLC system were kept in ambient temperature.

Structure of Tenofovir disoproxil, Emtricitabine and Efavirenz.

Tenofovir Disoproxil.

Emtricitabine

Efavirenz

Preparation of Mobile phase:

Sodium Dihydrogen Orthophosphate monohydrate (0.02M) buffer was prepared by dissolving 2.75 gms of buffer in 1000 ml of water and by adjusting the pH to 3.5 with dilute orthro phosphoric acid.

Fig 1:Typical Chromatogram of Emitricetabine (5.88mins), Tenofovir (8.796) and Efavirenz (12.015) by HPLC

Preparation of Standard Stock solution:

Accurately 40 mg of Emtricitabine, 60 mg of Tenofovir disoproxil fumerate and 120 mg of Efavirenz standards were weighed and taken in 25 ml volumetric flask. Dissolved by sonication in 15 ml of Diluent (in a ratio of 70:30 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.

Working Standard solution:

5 ml of the above standard stock solution was taken in 10 ml volumetric flask and made up to 10 ml with diluent to get a concentration of 800, 1200 and 2400 µg/ml for Emtricitabine, Tenofovir disproxil and Efavirenz respectively.

Preparation of Sample solution:

Ten tablets (Vireday, Cipla) were accurately weighed and crushed into a fine powder. The powder equivalent to one tablet (200 mg of Emtricitabine, 300 mg of Tenofovir disoproxil fumerate and 600 mg of Efavirenz) was taken in 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 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

Linearity:

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 Emtricitabine, Tenofovir disoproxil fumerate and Efavirenz respectively) were taken in different 10 ml volumetric flask and diluted up to the mark with Diluent. Evaluation was performed with photo diode detector (PDA) at 265 nm and Peak area was recorded for all the peaks and a Calibration graph was obtained by plotting peak area versus concentration of Emtricitabine (Fig 2 A), Tenofovir disoproxil fumerate (Fig 2 B) and Efavirenz respectively (Fig 2 C). The plot of peak area of each sample against respective concentration was found to be linear in the range of 8 - 120, 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 fumerate and Efavirenz respectively. Linear regression least square fit, slope (m), intercept (b), standard deviation, residual sum of squares and correlation coefficient data obtained from the measurements are given in Table I.

Table II: Results of HPLC assay and Recovery studies:

S. No.	Drug	Emtric itabine	Tenofovir Disoproxil fumerate	Efavirenz
1	Amount claim (mg/Tablet)	200	300	600
2	Amount Found (mg/Tablet)	199.56	300.07	602.05
3		198.64	299.22	600.33
4		198.64	299.90	600.69
5		198.63	299.38	601.22
6		198.12	298.71	601.50
7		198.07	298.57	601.32
Mean		198.61	299.31	601.19
8	*%Recovery	100.87	100.04	99.51

* Average of Three different Concentration levels.

Assay:

10 µl of sample solution was injected into liquid chromatograph. The retention time was found to be 5.875 mins for Emtricitabine, 8.800 mins for Tenofovir disoproxil fumerate and 12.020 mins for Efavirenz. The amount of drug present per tablet was calculated by comparing the respective peak areas of the sample solution with that of the standard solution. The data are presented in Table II.

Fig 2 B: Calibration curve of Tenofovir Disproxil by HPLC

Table III: Validation Summary: System Suitability as Per USP

Drug	Emtric itabine	Tenofovir Disoproxil fumerate	Efavirenz
Retention Time (mins)	5.875	8.800	12.020
Theoretical Plates (N)	5887	75254	64082
Tailing Factor	0.98	1.23	1.19
Capacity Factor (k')	-	0.498	1.046
Resolution	-	13.462	20.236
Relative Retention Time	1.00	1.50	2.05
Limit of Detection (µg/ml)	0.06	0.07	0.08
Limit of Quantification (µg/ml)	0.14	0.12	0.15

Recovery Studies:

Accuracy was determined by adding the known amount of drug substances of Emtricitabine, Tenofovir disoproxil fumerate and Efavirenz to the pre analyzed samples and subjected to the proposed HPLC analysis. Results of recovery study are shown in Table II. The study was done at three different concentration levels.

RESULTS AND DISCUSSION:

As per the USP-XXVI system suitability tests were carried out on freshly prepared standard stock solution of Emtricitabine, Tenofovir disoproxil fumerate and Efavirenz. Parameters that were studied to evaluate the suitability of the system are given in Table III. These parameters indicate good sensitivity, more ruggedness and robustness of the method.

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

The limit of detection (LOD) and limit of quantification (LOQ) for Emtricitabine, Tenofovir disoproxil fumerate and Efavirenz were found to be 0.06, 0.07 and 0.08 µg/ml and 0.14, 0.12 and 0.15µg/ml respectively. The signal to noise ratio is 3 for LOD and 10 for LOQ. From the typical chromatogram of Emtricitabine, Tenofovir disoproxil fumerate and Efavirenz as shown in fig 1, it was found that the retention times 5.875 mins for Emtricitabine, 8.800 mins for Tenofovir disoproxil fumerate and 12.020 mins for Efavirenz. A gradient program with mixture of buffer as mobile phase A (0.02M sodium dihydrogen orthro phosphate monohydrate) and methanol and water in a ratio of 85:15v/v as mobile phase B was found to be most suitable mobile phase combination to obtain well defined peaks with sharp peak shapes, high theoretical plates and less tailing. In the present developed HPLC method, the standard and sample preparation involve very simple extraction procedure and required very less time. A good linear relationship (more than r=0.999) was observed for Emitricitabine, Tenofovir Disoproxil fumerate and efavirenz in the concentration range of 8 - 120, 12 - 180 and 20 - 360 µg/ml respectively. The percentage assay was found to be 99.31% for Emtricitabine, 99.77 % for Tenofovir disoproxil fumerate and 100.20% for Efavirenz in tablets. Recovery studies shows good extraction and recovery from 50% to 150% of test 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

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.

REFERENCES:

1. S.Budhavari, The Merck Index (monograph#3521), 14, 598,(2006).

2. S.Budhavari, The Merck Index (monograph#3565), 14, 606,(2006).

3. S.Budhavari, The Merck Index(monograph#9146), 14, 1573(2006)

4. C.Sean Sweetman, Martindale-The Complete Drug Reference, 34,632, (2005).

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6. Indian pharmacopoeia vol.2, 1071, (2007).

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Received on 05.01.2009 Modified on 10.01.2009

Research J. Pharm. and Tech. 1(4): Oct.-Dec. 2008;Page 522-525

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