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Spectrophotometric Determination of Tenofovir Disoproxil Fumarate After Complexation With Ammonium Molybdate And Picric Acid

Johnson O. Onah* and Ukpe Ajima
Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, University of Jos, PMB 2084, Jos, Nigeria
 
Corresponding Author: Johnson O. Onah, Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, University of Jos, PMB 2084, Jos, Nigeria, E-mail: ogodaona@yahoo.co.uk
 
Received: 23 October 2010 Accepted: 25 January 2011
 
Citation: Johnson O. Onah and UkpeS Ajima "Spectrophotometric Determination Of Tenofovir Disoproxil Fumarate After Complexation With Ammonium Molybdate And Picric Acid", Int. J. Drug Dev. & Res., Jan-March 2011, 3(1): 199-204. doi: doi number
 
Copyright: © 2010 IJDDR, Johnson O. Onah et al. This is an open access paper distributed under the copyright agreement with Serials Publication, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
 
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Abstract

PURPOSE: Tenofovir disoproxil Fumarate (TDF) is a Nucleoside Reverse Transcriptase Inhibitor (NRTI) used in the management of HIV/AIDS. The drug has not been listed in the Official Pharmacopoeias and monographs. Two simple and sensitive methods for the determination of Tenofovir are described. METHODS: The first method involved the acid hydrolysis of the orthophosphate group of tenofovir disoproxil and its subsequent complexation with ammonium molybdate- Stannous Chloride to form a coloured complex with absorption maximum (ëmax) at 495nm. The second method involves complexation of the hydrolyzed tenofovir with picric acid to form a blue coloured complex that absorbed maximally (ëmax) at 465 nm. RESULTS: Both complexations obeyed Beer-Lamberts Law over the wide range of concentrations investigated. Complexation with ammonium molybdate-stannous Chloride and picric acid gave a mean recovery of 99.20% and 96.12% respectively. Their molar absorptivities were also calculated to be 1,234.09 Mol l-1cm-1 and 12,330.92 Mol l-1cm-1 respectively. The Sandell?s sensitivity were similarly determined as 1.94 and 19.40 respectively. Comparison of the two methods by Student?s t-test suggested that they are not significantly different (t ≥ 5 %). CONCLUSION: This results from this investigation show that both methods are suitable for evaluating the quality of the tenofovir disoproxil formulation and can be used for its routine quality control

Key words

 
Tenofovir disoproxil fumarate, Visible spectrophotometry, Ammonium molybdate, Charge transfer complexation, Picric acid.
 

Introduction

 
Tenofovir Disoproxil Fumarate (TDF) is an antiretroviral agent belonging to the class of nucleotide reverse transcriptase inhibitors [1].
 
Chemically TDF is 9-[(R)-2-[[ bis[(Isopropoxycarbonyl) oxy] methoxy] phosphinyl] methoxy] propyl] adenine fumarate. It is indicated in combination with other antiretroviral agents for the treatment of HIV-1 infection in adults [2]. It is a prodrug and so it is metabolized de novo to Tenofovir [3], an acyclic nucleoside phosphonate (nucleotide) analogue of Adenosine 5’-monophosphate. It is available in tablet dosage form only.
 
Although no assay procedure has been presented in any of the Official Pharmacopoeias, literature survey has revealed a number of analytical methods. These include liquid chromatography with tandem mass spectrometry [4, 5]; HPLC with solid phase extraction [6]; Reversed phase HPLC [7,8]; HPLC with spectrophotometric detection [9]; HPTLC [10]; Gradient ion-pair LC with fluorescent detector[11]; HPLC-UV and HPLC-MS [12] and Firstorder UV derivative spectrophotometry [13].
 
Charge transfer complexes (CTC) result from molecules that possess high occupied molecular orbital (HOMO) interacting with another molecule with low unoccupied molecular orbital (LUMO) by a donor-acceptor mechanism. For example, amines and other functional groups with excess electrons can engage in charge - transfer complexation reaction with electron acceptors such as Chloranil, Chloranilic acid and tetracyanoethylene [14,15,16,17]. Electron donors and acceptors may be classified into π or ä donors or acceptors. Quantitative analysis of many pharmaceutical and other nitrogenous compounds using CTC has been reported extensively in Literature [18,19,20, 21].
 
Some limitations of charge transfer complexation reactions in their application as an analytical technique include; the possible formation of termolecular and isomeric complexes, specific solvation of the donor and acceptor molecules and the empirical determination of the optical absorption due to the complex may be affected by absorption of the reactants themselves [22]. Also, there is a lack of direct evidence for the existence of the complex other than the colour formation.
 
Picric acid acts as a π acceptor although it has not been utilized as extensively as other piacceptors for the spectrophotometric determination of a variety of electron donating basic compounds. This paper presents two simple, precise and accurate methods for the spectrophotometric determination of TDF.
 

EXPERIMENTAL

 

Materials:

 
Tenofovir disoproxil fumarate tablets were kindly provided by Prof. J.A. Idoko, Director of Aids Prevention in Nigeria (APIN) Clinic, University of Jos Teaching Hospital, Jos Nigeria. The following reagents were manufactured by May & Baker, England: Ammonium Molybdate, Stannous Chloride. Similarly, hydrochloric acid, ethanol, chloroform, 1, 4-dioxan are all AnalaR grade (BDH, England). Picric acid (Riedel de Haem, Germany). All other reagents used were of analytical grade and were used without further purification.
 

METHOD:

 

Complexation with Ammonium Molybdate- Stannous Chloride Complex.

 
100 mg of the pure Tenofovir powder was dissolved in 100 mL of 3M Hydrochloric acid in a volumetric flask to form the stock solution of 1mg/mL. The solution was refluxed gently for 60 minutes to hydrolyze the phosphate groups from the drug. The hydrolyzed drug was partitioned with chloroform (25 ml x 4). Portions of the aqueous solution that now contain the phosphate ions were diluted to obtain the working concentrations of 5 µg/mL to 25 µg/mL.
 
Ammonium molybdate stock solution containing 1 mg/ml was prepared in water from which serial dilutions of 5 µg/ml to 25 µg/ml were similarly prepared. Into separate 10 ml volumetric flasks, 5 µg/ml to 25 µg/ml solutions of the hydrolyzed drug prepared above were quantitatively transferred followed by ammonium molybdate and 5 drops of stannous chloride. The volumes were brought up to mark with water. A blank solution was similarly prepared but without the drug. The flasks were allowed to stand for 30 minutes to allow for full development of colour. One of the solutions was scanned (Jenway Model 6405, England) to determine the absorption maximum.
 

Construction of the Calibration curve and its validation

 
Serial dilutions obtained as above were replicated in quadruplet. The flasks were thoroughly shaken and allowed to stand for 30 minutes before their absorbances were read on the spectrophotometer. The means of the absorbances were plotted against the concentrations using Microsoft Excel software program from which regression equation and coefficient were generated. Validation of the plot was carried out essentially by the same method except that various concentration ratios between phosphate and molybdate were used and then converted to the corresponding concentrations in µg using the calibration equations. A mathematical model equation that satisfied all the conditions of the experiment was generated.
 

Formation of Charge transfer complexes with Picric Acid

 
Solution of the drug in Chloroform obtained from the first part of this experiment was diluted to between 5 µg / ml to 25 µg /ml in separate 10 m volumetric flasks. Picric acid solutions equivalent to between 50 µg /ml to 250 µg /ml were similarly prepared. Equal volumes of picric acid and tenofovir (2 ml each) were quantitatively transferred into 10 ml volumetric flasks and the volumes made up to mark. The contents were allowed to stand for 20 minutes for colour to develop fully. The color formed was stable for over 60 minutes. A blank solution was similarly prepared. The absorption maximum was determined using the spectrophotometer.
 

Construction of the Calibration Curve and its validation

 
The absorbance and concentration of the volumetric flasks from above were read on the spectrophotometer (Jenway, model 6405, England). Three replicate preparations were similarly determined. Plots of absorbance against concentrations were generated using Microsoft Excel software program from which different equations and regression coefficients were obtained. The reproducibility, repeatability, accuracy, standard deviations were calculated and compared. The mathematical equation that satisfied the various conditions was generated thus validating the procedure.
 

Determination of tenofovir content by the developed methods:

 
Four different batches of tenofovir disoproxil were investigated and the following procedures were repeated in quadruplicate. Calibration curves were constructed for both methods as described above and different concentrations of tenofovir disoproxil were taken, treated to the procedures described above and their absorbances determined. The percentage recovery was then determined by comparing the concentrations obtained from the validated curves with the actual concentrations of the drug taken. Relative standard deviations were also calculated.
 

Data Analysis

 
The results, expressed as mean ± standard deviation were calculated using Microsoft Excel 2003 application
 

RESULT

 

Ammonium molybdate-Tenofovir complex

 
The complex formed between the molybdate and phosphate ion absorbed maximally at 495 nm. The plot of concentration of the drug against absorbance produced a linear curve with the following equation and regression coefficient:
 
Aabs = 1.901 * 10-3 x + 0 (Regression coefficient = 0.9989)
 
Where Aabs = absorbance and x the concentration component.
 
From the various plots obtained during the validation process, the standard deviations of the slope and intercept were 1.0 x 10-4 and 3.0 x 10-4 respectively. These results suggest that these measurements were quantitative, accurate and reproducible. Molar absorbtivity and Sandell’s sensitivity calculations were 1,234.09 Mol l-1cm-1 and 1.94 respectively. The content of tenofovir disproxil in a commercial formulation assayed was determined to be 99.20 % RSD ± 1.02 %
 
Picric acid- Tenofovir Complexation (CTC)
 
Picric acid in chloroform absorbs maximally at 400 nm. In the presence of tenofovir disoproxil, a bluepurple coloured complex was formed spontaneously with an absorption maximum at 465 nm. The complex formed obeyed Beer-Lamberts Law over a wide range of concentrations investigated. The following equation summarized the linearity between absorbance and concentrations determined. The regression coefficient was 0.9979.
 
Aabs = 0.0187x + 0
 
Where Aabs = absorbance and x represents the concentration factor. The standard deviation of the slope and intercept were calculated to be 2.0 x 10-3 and 1.0 x 10-4 respectively. The molar absorbtivity and Sandell’s sensitivity were calculated to be 12,330.92 Mol l-1cm-1 and 19.40 respectively. Quantitative determination of tenofovir disproxil by this technique gave recovery of 96.12 ± RSD 1.001 %.
 

DISCUSSION:

 

Ammonium molybdate - Tenofovir complexation

 
Ammonium molybdate is the standard reagent for the quantitative determination of phosphate groups in chemical substances. The acid hydrolysis of tenofovir disoproxil freed the phosphate group for this reaction to take place. The excellent recovery of 99.20% indicates the accuracy of this method. The Sandell’s figure further supports the sensitivity of this technique. Data obtained from Job’s continuous variation plot also shows that the stoichiometric relationship between the phosphate and molybdate was 1:1 and this is supported by the fact that the complex obeyed Beer-Lamberts Law. This observation also suggests that isomeric and termolecular complexes were not formed.
 
Picric acid- Tenofovir Complexation (CTC)
 
Picric acid is a π- acceptor while tenofovir disoproxil is a π- donor and the coloured complex formed is due to the donor-acceptor interaction between the two. Complex formation also led to the shift of the absorption maximum from 400nm to 465nm representing a bathochromic shift. The complex formed obeyed Beer-Lamberts Law over a wide range of concentrations investigated. The result presented here are in agreement with published reports on the nature of interaction between π-donors and acceptors [16], i.e. no isomeric complexes were formed. Observations from various published reports [15,19], have shown that absorption maxima range from 420 nm to 520 nm. This can be explained on the degree of the overlapping orbital occasioned by the stereochemistry of the interacting molecules. The Authors’ recent observation also suggests that the nature of the solvent affect the electronic transitions between π-acceptors and π-donors. The application of π-acceptors - π-donors interactions are proving themselves useful as analytical tool in pharmaceutical analysis.
 
Comparison of the two methods by Student’s t-test suggested that they are not significantly different (t ≥ 5 %).
 

CONCLUSION:

 
The new techniques developed for analysis of tenofovir disoproxil have proved successful because the procedures are simple, sensitive and significantly accurate. These techniques commend themselves to be considered for inclusion in official monographs.
 

Conflict of Interest

 
NIL
 

Source of Support

 
NONE
 

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