Spectroscopic Estimation of Tenofovir Alafenamide, an antiretroviral drug


Pankaj Kumar*, Jane Jacob, Khadjeeth Ajina, Jazeela, Fathima

Department of Pharmaceutical Chemistry, NGSM Institute of Pharmaceutical Science, NITTE University, Deralakatte, Mangalore-575018, Karnataka,India.

*Corresponding Author E-mail: pankajpgr@gmail.com





Received on 18.03.2016          Modified on 25.03.2016

Accepted on 08.04.2016        © RJPT All right reserved

Research J. Pharm. and Tech. 2016; 9(5): 538-540.

DOI: 10.5958/0974-360X.2016.00101.3




Tenofovir Alafenamide is a nucleotide reverse transcriptase inhibitor and a novel prodrug of tenofovir. A simple, economical and accurate UV spectroscopy method was developed for Tenofoviral Afenamide in methanol and distill water in the ratio 20:80. It obeyed Beer’s law in the linearity range of 10 -50 µg/ml at an absorption maxima of 267 nm. The results are reproducible with coefficient of variance less than 1%. The linear regression of absorbance on concentration with coefficient of regression of almost 1 indicates a good linearity between absorbance and concentration. The value of percentage relative standard deviation of less than 1% and low range of percentage error shows high degree precision and accuracy of the method.


KEYWORDS: Tenofovir Alafenamide, reverse transcriptase, tenofovir, HIV infection, UV spectroscopy.




Antiretroviral drugs which are used in management of HIV \AIDS normally includes the use of anti retro viral drugs in an attempt to control HIV infection1. There are several classes of anti retro viral agents that act on different stages of HIV life cycle .The use of multiple drugs that act on different viral targets is known as (HAART) highly active antiretroviral therapy2. Tenofovir Alafenamide is a nucleotide reverse transcriptase inhibitor and a novel prodrug of tenofovir. It is closely related to tenofovirdisoproxil fumarate but has greater antiviral activity. Mainly used in the treatment of HIV infection and chronic hepatitis B3. It is currently being studied as a three different investigational fixed dosage combination in the treatment of HIV infections.


A new simple, sensitive and specific procedure has been developed for determination of tenofovirdisoproxil fumarate in bulk and pharmaceutical dosage forms by Mannem SV et alusing MBTH reagent. 3-Methyl-2-benzothiazoline hydrazone reacts with the secondary amine group of tenofovir in the presence of oxidizing agent, ferric chloride. The resulting apple green coloured chromogen when measured spectrophotometrically in visible region (i.e., 400-800nm) shows a maximum absorbance at 626.5nm. This method can be successfully applied for the determination of drug content in pharmaceutical formulations4,5.


Tenofovirdisoproxil fumarate, Lamivudine and Efavirenz simultaneously, in a combined tablet form was estimated by Reverse Phase High Performance Liquid Chromatography (RP-HPLC). Symmetry C18 (4.6 x 100 mm, 3.5 µm) column operated with a mixture of phosphate buffer of pH 4.0 with ortho phosphoric acid and acetonitrile (42:58) as mobile phase was found to be suitable for the simultaneous estimation. The flow rate was maintained at 0.5 mL/min. Detection was carried out at 254 nm using a UV detector and the total run time was less than 11 minutes with the retention times of 2.2 min, 3.3 min and 10.8 min respectively for Lamivudine, Tenofovirdisoproxil fumarate and Efavirenz by Vanaja P et al6 .

A simple, rapid reversed-phase high performance liquid chromatographic method had been developed and validated for estimation of emtricitabine and tenofovirdisoproxil fumarate in tablet dosage form. The estimation was carried out on Luna C18 (25cm x 4.60 mm, particle size 5µm) column with a mixture of acetonitrile: potassium dihydrogen phosphate buffer (pH 3.0 ± 0.05 adjusted with orthophosphoric acid): triethylamine in the ratio of 70:30:0.5(v/v) as mobile phase. UV detection was performed at 260 nm. The method was validated for linearity, accuracy, precision, specificity and sensitivity as per ICH norms7.


A complexation, derivatization, extraction, evaporation, and sensitive-free direct UV spectrophotometric method is developed and validated by for the simultaneous estimation of some antiviral drugs such as emtricitabine (EMT), tenofovirdisoproxil fumarate (TDF), and rilpivirine HCl (RPV) in tablet dosage form by Vierordt’s method. The solutions of standard and sample were prepared in methanol. The foremtricitabine, tenofovirdisoproxil fumarate, and rilpivirine hydrochloride were 240.8 nm, 257.6nm, and 305.6 nm, respectively. Calibration curves are linear in the concentration ranges 4–12 μg/ml for EMT, 6–18 μg/ml for TDF, and 0.5–1.5 μg/ml for RPV, respectively. Results of analysis of simultaneous equation method were analyzed and validated for various parameters according to ICH guidelines8,9 .



All the solvents used were of AR grade and the spectral measurements were carried out on Shimadzu UV/VIS 1700 spectrophotometer with the help of a pair of 1cm matched quartz cuvettes of 10 mm optical path length. Tenofoviral Afenamide was obtained as a gift sample from Mylan Laboratories, Hyderabad.


The stock solution of tenofovir was prepared by accurately weighing 100 mg of pure drug into a 100ml standard volumetric flask then dissolved in 20 ml of methanol and made to the required volume with distilled water.


A second stock solution was prepared by diluting 5ml of solution from the first stock solution and into a 100ml volumetric flask and the volume is made upto 100ml with distill water.


From the second stock solution, working solution was prepared so as to get a concentration of 50µg/ml and absorption maxima of the solution was determined by scanning in the region between 200-400 nm. The absorption maxima was found to be 267 nm. The stability of the compound in the solvent was carried out and it is found to be stable for more than 6 hrs.

Aliquots of drug solution of 2 - 10 ml were pipetted out into 5 standard volumetric flask of capacity 10ml each and the volume was made up to the 10 ml mark with distill water. The absorbance of these solutions was measured at 267 nm against a blank solution and the calibration curve plotted.



The developed method was validated as per ICH guidelines. The optical characteristics such as Beer’s law limit, molar absorptivity and other parameters for the proposed method are summarized in Table I. six trials for each concentration were carried out to check the linearity (Fig 2) and eight trials were carried out to determine the relative standard deviation. The linear regression of absorbance on concentration with a correlation coefficient (r) of almost 1 indicates a good linearity between absorbance and concentration .The value of percentage relative standard deviation less than 1% and low percentage range of error confirm the high degree of precision and accuracy of the proposed method.


Table 1: Optical characteristics and Precision



λ max

267 nm

Beers law limits (µg/ml)


Molar absorptivity (l/mol.cm)

7.22 x 104

Sandell’s sensitivity (µg/cm2/0.001 AU)


% Relative Standard Deviation


Correlation coefficient


Regression equation (y=a+bc)


      Intercept (a)



5.29 x 10-3

Confidence limits

      0.05 level

      0.01 level




% Range of error

      0.05 level

      0.01 level






The developed method is simple, economical and accurate therefore can be employed for routine laboratory analysis of tenofoviral Afenamide.



The authors are thankful to Mylan Laboratories, Hyderabad for providing Tenofoviral Afenamide as gift sample and  Nitte University for providing the fund and necessary facilities to carry out this research



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