INTRODUCTION:

Emtricitabine (4-amino-5-fluoro-1-[(2S,5R)-2-(hydroxymethyl)-1,3-oxathiolan-5-yl]-1,2-dihydropyrimidin-2-one), an analogue of cytidine, and Tenofovir disoproxil fumarate (({[(2R)-1-(6-amino-9H-purin-9-yl)propan-2-yl]oxy}methyl)phosphonic acid), are nucleoside reverse transcriptase inhibitor (NRTI) that are used for the treatment of HIV infection.¹

Fig: Chemical Structure of Emtricitabine

Tenofovir alafenamide fumarate (TAF) is a nucleotide reverse transcriptase inhibitor (NRTI) and a novel ester prodrug of the antiretroviral tenofovir. Following oral administration, TAF is converted in vivo to tenofovir, an acyclic nucleoside phosphonate (nucleotide) analog of adenosine 5′-monophosphate. Tenofovir mimics normal DNA building blocks but is lacking a 3'-OH molecule required for phosphodiester bond linkage. By competing with regular nucleotides for incorporation into proviral DNA and prevention of the formation of the 5' to 3' phosphodiester linkage required for DNA elongation, tenofovir causes early chain termination and prevents proviral DNA transcription.

Although tenofovir (available as tenofovir disoproxil fumarate) has a good safety profile and efficacy, and is currently a cornerstone of HIV antiviral treatment, its use has been associated with nephrotoxicity and reduced bone mineral density.²

Fig: Chemical Structure of Tenofovir Alafenamide fumarate

A literature survey revealed that UV spectroscopic techniques , liquid chromatography techniques have been reported for the simultaneous determination of emtricitabine, and tenofovir disoproxil fumarate in pure drug. But no Spectrophotometric or Chromatogrphic Method was reported for analysis of emtricitabine, and tenofovir alafenamide fumarate. Hence, the authors have attempted to develop a simple, rapid, precise and accurate method for the simultaneous estimation of these drugs in Pure Drugs. ^3-9

MATERIAL AND METHODS:

Chemicals and Reagents:

Pharmaceutical grade of Emtricitabine and Tenofovir Alafenamide Fumarate were kindly supplied as gift sample by Lupin. Distilled water was used as solvent.

Instruments:

UV-Visible Spectrophotometer-Agillent with cuvette cells of one cm light path were used for the measurement of absorbance. Electronic Balance of Schimadzu BL-220H was used for weighing the samples. Class ‘A’ volumetric glassware were used. UltraSonicator- wensar was used for sonication purpose.

Procedure:

UV Spectrometric methods for determination of Emtricitabine and Tenofovir Alafenamide fumarate:

Selection of Solvent:

The solvent was selected by determining the solubility of Emtricitabine and tenofovir Disproxil fumarate in various solvents namely Distilled water, Hydrochloric Acid, Sodium Hydroxide Solution, Methanol. Finally, Distilled water was chosen as the solvent for Emtricitabine and Tenofovir alafenamide fumarate depending on absorption at its analytical wavelength.

Preparation of standard stock solution:

An accurately weighed quantity of about 10mg Emtricitabine and Tenofovir Alafenamide fumarate was taken in a 100 mL volumetric flask and was dissolved in Distilled wtaer with sonication. The volume was made upto mark with Distilled water to get the concentration of 100ppm.

Preparation of Working Standard Solution of Emtricitabine and tenofovir Alafenamide fumarate :

The aliquot portion of standard stock solution of Emtricitabine and Tenofovir Alafenamide fumarate was diluted appropriately with Distilled water obtaining concentration 10 μg/mL. Solution was taken in 1 cm cell and scanned in the range 200 nm to 400 nm and spectrum was recorder as showed in Fig 1

Fig1: UV Spectrum of Emtricitabine

Fig 2: UV Spectrum of Tenofovir Alafenamide Fumarate

Fig 3: Overlain Spectra of Emtricitabine And Tenofovir Alafemanide Fumarate

Method Validation:

Specificity

Specificity was studied by measuring the absorbance of EMT and TAF individually at 280 nm and 260 nm against the blank and comparing the absorbance of drugs solutions to the blank. No interference was observed.

Linearity and range:

Five aliquots of each drug solutions were taken from standard stock solution and transferred to 10ml volumetric flask to get a final concentration of 5, 10, 15, 20, 25 and 30 μg/ml of Emitrictabine and 5, 10, 15, 20, 25 and 30 μg/ml of Tenofovir Alafenamide Fumarate and the volume was made upto the mark with the distilled water and each flask content was measured to determine the absorbance at the selected wavelength. For simultaneous equation method the absorbance of all standard solutions were measured at 260nm and 280nm, the calibration curves of absorbance vs. concentration was plotted and correlation coefficient and regression line equations for both EMT and TDF were determined. Straight line equations were obtained from these calibration curves. The linear regression equation of Emtricitabine y=0.0351x-0.0671 (R² = 0.9972) and Tenofovir Disoproxil Fumarate was y = 0.0252x +0.0140 R² = 0.9999. The result were reported in Table No 1and 2.

Limit of Detection and Limit of Quantification :

ICH guideline describes several approaches to determine the detection and quantification limits. These include visual evaluation, signal-to-noise ratio and the use of standard deviation of the response and the slope of the calibration curve. In the present study, the LOD and LOQ were based on the third approach and were calculated according to the 3.3 × (SD/Slope) and 10 × (SD/Slope) criteria, respectively; where SD is the standard deviation of y-intercept of regression line and S is the slope of the calibration curve.

Application of the proposed procedures for the simultaneous determination of Emtricitabine and tenofovir Alafenamide fumarate in laboratory prepared mixtures

Method A: Simultaneous Equation Method:

Mixtures of the two drugs were prepared by diluting 1.0 mL of 100ppm stock solution of Emtricitabine and Tenofovir Alafenamide fumarate with distilled water upto 10 mL. The concentrations of both Emtricitabine and Tenofovir Alafenamide fumarate were determined by measuring the absorbance of the prepared mixtures at 280 nm and 260 nm. From these absorbance values, the concentrations of EMT and TEN were determined using Simultaneous equation method.

The absorptivity of each solution was calculated by using the following formula:

Absorptivity=Absorbance / concentration (gm/100 mL)

The absorbance of the sample (formulation) at λ₁(280) and λ₂(260) is A₁and A₂ respectively.

The total absorbance of the mixture is equal to the sum of individual absorbance of X and Y.

A₁ = ax₁bCx + ay₁bCy

A₂ = ax₂bCx + ay₂bCy

Cx= A₂ay₁-A₁ay₂/ ax₂ ay₁- ax₁ ay₂

Cy=A₁ax₂-A₂ax₁/ ax₂ ay₁- ax₁ ay₂

Cx – concentration of EMT

Cy – concentration of TEN

Method B- Q – Absorbance ratio Method:

This method is applicable to the drugs that obey Beer’s law at all wavelengths and the ratio of

absorbance at any two wavelengths is a constant value, independent of concentration and path length.The solutions of 8μg/ml and 10μg/ml for Emtricitabine and Tenofovir alafenamide Fumarate were scanned in the wavelength range of 400 to 200nm to obtain overlain spectra (fig 5). Two wavelengths, 251nm as isoabsorptive point and 237nm (λmax of Emtricitabine) were selected for the formation of Q absorbance ratio equation. Mixtures of the two drugs were prepared by diluting 1.5 mL and 2.0 ml of Emtricitabine and Tenofovir Alafenamide fumarate from100ppm stock solution and diluted upto 10 mL with distilled water.The absorptivity coefficients of each drug at both the wavelengths were determined by using 10ug/mL solution of Emtricitabine and Tenofovir alafenamide fumarate. The concentration of the individual components, Cx and Cy can be calculated by using the following equations.

Cx = (QM-QY/QX-QY) x (A1/ax1)

CY = (QM-QX/QY-QX) x (A2/ay1)

Where A1 and A2 are absorbance of sample solution at iso-absorptive point 251 nm and 237 nm λmax of Emtricitabine respectively, ax1 and ax2 are the absorptivities of the Emtricitabine at 237nm and 251nm respectively and ay1 and ay2 are the absorptivities of the Tenofovir Alafenamide Fumarate at 237nm and 251nm respectively.

RESULTS AND DISCUSSION:

A reliable Simultaneous Equation method and Q- method was developed for simultaneous estimation of Emtricitabine and Tenofovir alafenamide Fumarate in bulk by UV Spectrophotometry. Beers law was obeyed in concentration range of 5-30 μg/ml for Emtricitabine and 5-30 μg/ml for Tenofovir alafenamide Fumarate at 280 nm and 260 nm wavelengths, respectively. The correlation coefficient Emtricitabine and Tenofovir alafenamide Fumarate was found to be R²= 0.9972 and 0.9993 respectively. The LOD and LOQ were 1.43 μg/mL and 4.33 μg/mL for Emtricitabine and 1.13 μg/mL and 3.43 μg/mL of Tenofovir Alafenamide Fumarate, respectively.

Table No.1 --Calibration data forEmtricitabine

Sr.No	Concentration in ppm	Absorbance
1	5	0.228
2	10	0.446
3	15	0.575
4	20	0.781
5	25	0.938
6	30	1.120

Fig 4: Calibration Graph of Emtricitabine

Table No. 2 -Calibration data for Tenofovir Disoproxil Fumarate

Sr.No	Concentration in ppm	Absorbance
1	5	0.1698
2	10	0.3520
3	15	0.5210
4	20	0.7052
5	25	0.8720
6	30	1.0256

Fig 5: Calibration Graph of Tenofovir alafenamide Fumarate

Method A: Simultaneous Equation Method:

Table 3: Data for Simultaneous Equation method

Name	Absorbance at 260nm	Absorptivity	Absorbance at 280 nm	Absorptivity
10ppm of Emtricitabine	0.3055	305.5 (ax₁)	0.4315	431.5 (ax₂)
10ppm of Tenofovir Alafenamide Fumarate	0.3514	351.4 (ay₁)	0.0818	81.8 (ay₂)
Lab Mixture	0.6561 (A1)		0.5133(A2)

The Equation becomes

0.6561= 305.5bCx + 351.4bCy

0.5133= 431.5bCx + 81.8bCy

Table 4: % recovery of Emtricitabine and Tenofovir Alafenamide Fumarate by Simultaneous Equation Method

Sr. No

Concentration of EMI Taken

Concentration of THF Taken

Concentration of EMI found

Concentration of THF found

% recovery of EMI

% Recovery of THF

10ug/mL

10 ug/mL

10.01 ug/mL

9.97ug/mL

100.01%

99.7%

Method B: Q Absorbance ratio method

Table 5: Data for Q method

Name	Absorbance at 237 nm	Absorptivity	Absorbance at 251 nm	Absorptivity
10ppm of Emtricitabine	0.4403	440.3 (ax₁)	0.3432	343.2 (ax₂)
10ppm of Tenofovir Alafenamide Fumarate	0.1633	163.3 (ay₁)	0.2831	283.1(ay₂)
Lab Mixture	0.9909(A1)		1.0872(A2)

Table 6: % recovery of Emtricitabine and Tenofovir Alafenamide Fumarate by Q- Method

Sr.No	Concentration of EMI Taken	Concentration of THF Taken	Concentration of EMI found	Concentration of THF found	% recovery of EMI	% recovery of THF
1	15ug/mL	20 ug/mL	15.01 ug/mL	20.20ug/mL	100.07%	101.03%

CONCLUSION:

Two methods i.e. Simultaneous equation method and Q.method has been developed for analysis of Emtricitabine and Tenofovir Alafenamide fumarate in Combination. The developed method was found to be Simple and economical, can be used for the routine Quality Control analysis of both the drugs in combined dosage form.

ACKNOWLEDGEMENT:

The authors thank Dr. A.G. Jadhav principal Sandip Institute of Pharmaceutical Sciences for providing all the facilities for Research work. They also thank to Lupin Pharma, India for providing standard .

REFERENCES:

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

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

3. Shinde M, Shelke A., Mogal R., Sable R., Jadhav A. Development And Validation of UV Spectrophotometeric method For Simultaneous Estimation Of Emtricitabine And Tenofovir Disproxil Fumarate In Bulk And Tablet Formulation By Simultaneous Equation Method.J Global Trends Pharm Sci, 2017; 8(1): 3614-3621.

4. Deepthi Komaroju, G. Nagarjuna Reddy, K. Dhanalakshmi "Method Development And Validation For Simultaneous Estimation Of Emtricitabine And Tenofovir Disoproxil Fumarate In Pure And Tablet Dosage Form By Using Rp-Hplc" International Journal Of Pharma Research and Review, Oct 2013; 2(10):1-11.

5. Swapnil A Ghorpade, Monali S. Sali, Atul H. Kategaonkar, Dhaval M. Patel, Vishnu P Choudhari, Bhanudas S Kuchekar "Simultaneous Determination Of Emtricitabine And Tenofovir By Area Under Curve And Dual Wavelength Spectrophotometric Method" J. Chil. Chem. Soc., 55, Nº 1 (2010).

6. Bala Rami Reddy.Yenumula, Mutta Reddy.Singampalli and Bala Sekhara Reddy.Challa "Simultaneous Estimation Of Emtricitabine And Tenofovir Disoproxil Fumarate In Tablet Dosage Form By Reverse Phase High-Performance Liquid Chromatography"Soj Chromatograph Sci 1(1): 6.

7. Maithilee Joshi, A. P. Nikalje, M. Shahed, M. Dehghan "Hptlc Method For The Simultaneous Estimation Of Emtricitabine And Tenofovir In Tablet Dosage Form" Indian J. Pharm. Sci., 2009, 71 (1): 95-97.

8. International Conference On Harmonisation Guideline On Validation Of Analytical Procedures: Text And Methodology; Q2 (R1), 2005

9. A.H.Becket And J.B.Stenlak, Practical Pharmaceutical Chemistry, 4th Edition Part- 2,CBS Publishers and distributors, 284-285.

Received on 25.04.2018 Accepted on 14.06.2018

Asian J. Pharm. Tech. 2018; 8 (2):103-107 .

DOI: 10.5958/2231-5713.2018.00016.8