UV-Spectrophotometric Absorbance Correction Methods for the Simultaneous Estimation of Tenofovir Alafenamide and Emtricitabine in Bulk and Tablet Dosage Form

 

Murugan S^1,2*, Vetrichelvan T¹

¹Department of Pharmaceutical Analysis, Adhiparasakthi College of Pharmacy, Melmaruvathur-603319, Tamilnadu, India.

²Research Scholar, The Tamilnadu Dr. M.G.R. Medical University, Chennai-600032, Tamilnadu, India.

 

ABSTRACT:

Two UV-spectrophotometric methods have been developed and validated for simultaneous estimation of Tenofovir alafenamide (TAF) and Emtricitabine (EMT) in bulk and tablet dosage form. The Method A employs absorption correction method in zero order, the wavelengths selected were 261.5 nm for TAF (but EMT shows absorbance) and 302 nm for EMT. Method B employs, absorption correction method in first order, the wavelengths selected were 218.5 nm for TAF (but EMT shows absorbance) and 300 nm for EMT. For both the methods drugs were linear between the range of 2-10 μg/ml for TAF and 16-80 μg/ml for EMT using methanol as solvent. The correlation coefficient was found to be 0.9996 for TAF and 0.9997 for EMT for method A and 0.9998 for TAF and 0.9999 for EMT for method B, respectively. The precision (intraday, interday) of methods was found within limits (RSD < 2%). It could be concluded from the results obtained in the present investigation that the two methods for simultaneous estimation of TAF and EMT in bulk and tablet dosage form are simple, rapid, accurate, precise and economical and can be used, successfully, in the quality control of tablet formulations and other routine laboratory analysis.

 

 

INTRODUCTION:

Tenofovir alafenamide (TAF) (Fig.1) is chemically isopropyl (2S)-2-[[[(1R)-2-(6-aminopurin-9-yl)-1- methyl-ethhoxy]methyl-phenoxy-phoshoryl] amino] propanoate. TAF is a nucleotide reverse transcriptase inhibitor and a prodrug of tenofovir. It is used in the treatment of HIV infection and chronic hepatitis B. TAF has greater antiviral activity and better distribution into lymphoid tissues than that tenofovir disproxil. Emtricitabine (EMT) (Fig.2) is chemically 4-amino-5-fluoro-1-[(2R,5S)-2 (hydroxyl -methyl)-1,3-oxathiolan-5-yl]-1,2-dihydro- pyrimidin-2-one. EMT is a nucleoside reverse transcriptase inhibitor (NRTI) for the treatment of HIV infection in adults. EMT is an analogue of cytidine.

 

The drug works by inhibiting reverse transcriptase, the enzyme that copies HIV RNA into new viral DNA. On literature survey, several methods were reported for the estimation of TAF and EMT individually [1-12] and in combination with other drugs[13]. So we have developed a novel, simple, rapid, accurate, precise, economical and highly sensitive UV spectrophotometric methods for assay of TAF and EMT in bulk and tablet dosage form and validated according to ICH guidelines.

 

 

Fig. 1 Chemical structure of TAF

Fig. 2 Chemical structure of EMT

 

 

MATERIALS AND METHODS:

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 TAF and EMT was procured from Beijing mesochem technology co.ltd. (China). The pharmaceutical dosage form used in this study was a Tafero EM tablets manufactured by Hetero Labs Limited, Himachal pradesh labeled to contains 200 mg of EMT and 25 mg of TAF was purchased from Median medex, Pune.

 

Preparation of stock standard solutions:

Stock standard solution of TAF (250 𝜇g/ml) was prepared by dissolving 25 mg of TAF in 100ml of Methanol in 100mL volumetric flask with vigorous shaking. From this stock standard solution, Similarly, stock standard solution of EMT (2000 𝜇g/mL) was prepared by dissolving 200mg of EMT in 100ml of methanol in 100ml volumetric flask with vigorous shaking.

 

Methods:

Method A: Absorption correction method in zero order:

In this method, solutions of TAF and EMT (10 μg/ ml, each), were prepared separately by appropriate dilution of standard stock solution with Methanol and scanned in the spectrum mode from 200 to 400 nm. From the overlay spectra [Fig.3] of TAF and EMT, two wavelengths were selected, one at 261.5 nm (λ2) for TAF and the other at 302 nm (λ1) for EMT at which TAF shows zero absorbance.

The absorbances of the sample solutions were measured for both the drugs at selected wavelengths. The concentrations of drugs in sample solution were determined by using the following formula:

 

 

Where,

C_x= Conc of EMT

C _y= Conc. of TAF

A₁= Absorbance of Sample Solution at λ₁ (302 nm)

A ₂=Absorbance of Sample Solution at λ₂ (261.5 nm)

ax₂, ay₂= Absorptivities of TAF and EMT at λ₂ (261.5 nm)

ax ₁ = Absorptivity of EMT λ₁ (302 nm)

 

Absorption correction method in first order:

Method B: Absorption correction method in first order:

In this method, solutions of TAF and EMT (10 μg/ ml, each), were prepared separately by appropriate dilution of standard stock solution with distilled water and scanned in the spectrum mode from 200 to 400 nm. The absorption spectra thus obtained were derivatized for first order. From the overlay first order derivative spectra [Fig.4] of TAF and EMT, two wavelengths were selected, one at 218.5 nm (λ2) for TAF and the other at 300 nm (λ1) for EMT at which TAF shows zero absorbance.

 

 

Fig.3. The overlain UV absorbance spectrum of TAF and EMT

 

Fig.No.4. The overlain first order derivative spectra of TAF and EMT

 

The absorbances of the sample solutions were measured for both the drugs at selected wavelengths. The concentrations of drugs in sample solution were determined by using the following formula:

                 A₁

C_x  = --------             

                ax ₁

               A _{2   -(} C_x ax_{2 )}

C _y =-------------------

             ay₂

 

Where,

C_x= Conc of EMT

C _y= Conc. of TAF

A₁= Absorbance of Sample Solution at λ₁ (300 nm)

A ₂=Absorbance of Sample Solution at λ₂ (218.5 nm)

ax₂, ay₂= Absorptivities of TAF and EMT at λ₂ (218.5 nm)

ax ₁ = Absorptivity of EMT λ₁ (300 nm)

 

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 200 mg of EMT was transferred to 100 ml volumetric flask; 50 ml methanol was added and sonicated for 15 min, volume was then made up to the mark with methanol. The resulting solution was mixed and filtered through whatmann filter paper no 41 and filtrated. 1.6 ml was withdrawn and diluted to 100mL using methanol to get 32 𝜇g/ml of Emtricitabine and 4 μg/ ml of TAF, the concentration of TAF and EMT were determined by measuring absorbance of sample solution in absorption correction method for zero order were 261.5 nm for TAF and 302 nm for EMT. In absorption correction method for first order, the wavelengths selected were 218.5 nm for TAF and 300 nm for EMT. Concentration of TAF and EMT in the diluted solution was obtained from calibration curves. Amount of TAF and EMT in mg/tab was then calculated, by multiplying the concentration obtained with dilution factor.

Validation:

The proposed methods were validated as per ICH guidelines.

 

Linearity:

Different aliquots of 0.8-4.0 ml of TAF were transferred into series of 100 ml volumetric flasks, separately and the volume was made up to the mark with Methanol to get concentrations 2, 4, 6, 8 and 10μg/ml, respectively. Similarly, different aliquots of 0.8-4.0 ml of EMT were transferred into series of 100ml volumetric flasks, separately and the volume was made up to the mark with Methanol to get concentrations 16, 32, 48, 64 and 80μg/ml. The absorption spectrum was recorded at 261.5 nm for TAF and EMT and also at 302.0 nm for EMT in absorption correction method for zero order. The absorption spectrum were derivatized to get first order and recorded by measuring absorbance in first order for absorption correction method derivative at 218.5 nm for TAF and EMT and also at 300.0 nm for EMT, respectively.

 

Accuracy:

To the preanalysed sample solutions, a known amount of standard stock solution was added at different levels i.e. 50, 100 and 150 %. The solutions were reanalyzed by proposed method.

 

Precision:

The reproducibility of this 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).

 

Table 1. Results of Analysis of Tablets

Tablet sample	Label claim (mg/tab)	% Label claim (n = 6)		%RSD
		Method 1	Method 2	Method 1	Method 2
TAF	25	99.28	99.94	0.4536	0.5831
EMT	200	100.32	100.12	0.5532	0.6322

 

Table 2. Results of Recovery Studies

Recovery level	Initial amount (𝜇g/mL)		Concentration of std drug added (𝜇g/mL)		%Recovery (𝑛 = 3)
					Method 1		Method 2
	TAF	EMT	TAF	EMT	TAF	EMT	TAF	EMT
50%	4.0	32.0	2.0	16.0	99.63	99.62	99.61	99.83
100%	4.0	32.0	4.0	32.0	99.76	100.14	99.76	100.32
150%	4.0	32.0	6.0	48.0	100.32	100.84	100.12	100.63
			Mean		99.90	100.20	99.83	100.26

 

RESULT AND DISCUSSION:

The methods discussed in the present work provide a convenient and reliable way for quantitative determination of TAF and EMT in combined dose tablet formulation.  The wavelengths selected for the absorption correction method in zero order were 261.5 nm for TAF (but EMT shows absorbance) and 302 nm for EMT.

 

In absorption correction method for first order, the wavelengths selected were 218.5 nm for TAF (but EMT shows absorbance) and 300 nm for EMT. Percent label claim for TAF and EMT in tablet analysis was found in the range of 99.28 to 100.32 % in absorption correction method for zero order and 99.94 to 100.12 % in absorption correction method for first order as shown in Table 1. Percent recovery for TAF and EMT, was found in the range of 99.90 to 100.20 % in absorption correction method for zero order and 99.83 to 100.26 % in absorption correction method for first order with standard deviation well below 2 indicating accuracy of this methods as shown in Table 2. 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 methods.

 

CONCLUSION:

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

 

ACKNOWLEDGMENT:

The management of ACMEC trust is hereby thanked by one of the author Mr.S.Murugan for their support in doing this research.

 

 

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Received on 19.03.2019           Modified on 20.04.2019

Accepted on 18.05.2019         © RJPT All right reserved