Estimation of Tenofovir Disproxil Fumarate in Bulk and Formulations by Visible Spectrophotometric Methods

 

Dr. Kalyana Ramu Buridi*

Department of Chemistry, Maharajah’s College (Aided & Autonomous), Vizianagaram-535002, Andhra Pradesh (India)

 

 

ABSTRACT:

Two direct, simple and sensitive visible spectrophotometric methods (A and B) have been developed for the determination of tenofovir disproxil fumarate in bulk and tablet dosage forms.  The method A is based on the reaction of drug with aromatic aldehyde such as Para dimethyl amino cinnamaldehyde (PDAC) in the presence of sulphuric acid in non aqueous medium and formed purple red colored condensation products with an absorption maximum of 502nm. The method B is based on the reaction of the drug with nitrous acid for diazotization and followed by coupling with N-(1-Naphthyl) Ethylene Diamine dihydrochloride (NED) in acid medium to form colored species with wavelength maximum at 500nm. The Beer’s law obeyed in the concentration range of 20-60 µg/ml, 8-24 µg/ml for methods A and B respectively. The proposed methods are applied to commercial available tablets and the results are statistically compared with those obtained by the reference UV method and validated by recovery studies.

 

KEYWORDS: Assay, Beer’s Law, Condensation reaction, Coupling reaction, PDAC, Regression equation

 

INTRODUCTION:

Tenofovir Disoproxil Fumarate (TDF) (Fig.1) is an antiretroviral agent belonging to the class of nucleotide reverse transcriptase inhibitors (NRTI) used in the management of HIV infection in adults. It is an orally bio available prodrug of tenofovir and the first nucleotide analogue approved for HIV-1 treatment1-2. In vivo, TDF is converted to tenofovir, an acyclic nucleoside phosphonate (nucleotide) analog of adenosine 5’-monophosphate. Tenofovir exhibits activity against HIV-1, HIV-2 and hepatitis-B virus. Chemically it is the 1:1 salt of the bis-isopropyloxy carbonyl oxy methyl ester of tenofovir and fumaric acid [9-[(R)-2-[[bis [(isopropoxycarbonyl) oxy] methyl] phosphinyl] methoxy] propyl] adenine fumarate]. Its empirical formula is C19H30N5O10P.C4H4O4   representing   molecular weight of 635.52. It is a white to off-white crystalline powder with a solubility of 13.4mg/ml in distilled water and freely soluble in methanol and in DMF. The drug is available in tablet dosage forms only. TDF remains in cells for longer periods of time than many other antiretroviral drugs, thereby allowing for once-daily dosing. The drug is official in IP3.

 

 

Figure 1: Chemical structure of tenofovir disproxil fumarate

 

Before phosphorylation, TDF is converted to tenofovir in the intestinal lumen and plasma by diester hydrolysis. Tenfovir then internalized into cells, possibly by endocytosis, and subsequently phosphorylated in sequential steps to tenfovir monophosphate and to the active metabolite, tenfovir diphosphate. In a mechanism similar to that of NRTI’s, tenfovir diphosphate competes with its natural nucleotide counterpart deoxyadenosine5’-triphosphate, for incorporation into newly forming HIV DNA. Once successfully incorporated, termination of the elongating DNA chain ensues, and DNA synthesis is interrupted.

 

TDF has been well tolerated in clinical trials with duration of follow-up to 96 weeks. It is associated with more favorable lipid profiles than stavudine and has not been associated with the mitochondrial toxicity attributed to other nucleoside analogues.

 

Extensive literature review reveals that several spctrophotometric (UV and Visible)4-15, HPLC16-24, HPTLC25-26 and LC/LC-MS27-33 methods have been reported so far for determination of tenofovir alone and its combination with other drugs. Even though some visible spectrophotometric assay procedures have been reported for the determination of TDF, many of them concern with biological fluid samples and very few in pharmaceutical formulations. Hence it is felt necessary to develop suitable visible spectrophotometric methods for the assay of TDF in both bulk drug and pharmaceutical formulations. So the author has made some attempts in this direction and succeeded in developing two methods based on the reaction between the drug and aromatic aldehydes such as PDAC34 in the presence of sulphuric acid in non aqueous medium or treating the drug with HNO2 and followed by coupling with NED to form purple colored species and stable for 30 minutes. The proposed methods for TDF determination have many advantages over other analytical methods due to its rapidity, normal cost and environmental safety. Unlike HPLC, HPTLC procedures, the instrument is simple and is not costly. Economically, all the analytical reagents are inexpensive and available in any analytical laboratory. These methods can be extended for the routine quality control analysis of pharmaceutical products containing TDF.

MATERIALS AND METHODS:

Apparatus and chemicals:

A Shimadzu UV-Visible spectrophotometer 1601 with10mm matched quartz cells was used for all spectral measurements. A Systronics digital pH meter mode-361 was used for pH measurements. All the chemicals used were of analytical grade.  PDAC (E. Merck, 0.1% w/v 6.31x 10-3M) in methanol and Sulphuric acid (14M), Sodium nitrite solution (Prepared by dissolving 250mg of NaNO2 in 100ml distilled water), 2.5%aqueous solution of ammonium sulphamate, 3M aqueous solution of sodium acetate and  0.1%NED solution (Prepared by dissolving 100mg NED in 100ml distilled water) were used for methods A and B respectively. 

 

Preparation of Bulk and Sample solution:

About 100mg of TDF [pure or formulation] was accurately weighed and dissolved in  100ml 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 (25ml x4). The chloroform extract was evaporated to dryness and the residue so obtained was dissolved in 100 ml methanol. The working standard solution of TDF (200 µg/ml) was obtained by appropriately diluting the standard stock solution with the same solvent. The prepared stock solution was stored at 4⁰ C protected from light. From this stock solution, a series of standards were freshly prepared during the analysis day.

 

Analytical Procedure: 

Determination of wavelength maximum (λ max):

Method A:

The 3.0 ml of working standard solution of TDF (200µg/ml) in methanol was taken in 10ml standard flask and volume of test tube adjusted to 3.0ml with methanol. To test tube 1.0 ml of PDAC (6.31x 10-3M) and 1.0 ml of concentrated sulphuric acid (14M) were added, while cooling under a tap with constant shaking and kept in water bath at 60şc for 10min for complete color development. Then cooled and diluted to the mark with methanol. In order to investigate the wavelength maximum, the above colored solution was scanned in the range of 360-560nm by UV-Visible spectrophotometer. From the spectra (Fig.2), it was concluded that 502nm is the most appropriate wavelength for analyzing TDF with suitable sensitivity.

 

Method B:

To 3.0ml of neutral solution of drug, 1.0ml of 2N HCl and 1.0ml of NaNO2 solutions were added successively into 25 ml calibrated tubes and kept aside for 15 min. after that 1.0ml of 2.5% aqueous solution of ammonium sulfa mate, 2.0ml 3M aqueous solution of sodium acetate and 1.0ml of 0.1% aqueous NED solutions were added successively. Purple colored species was formed. In order to investigate the wavelength maximum, the above colored solution was scanned in the range of 360-560nm by UV-Visible spectrophotometer. From the spectra (Fig.3), it was concluded that 500nm is the most appropriate wavelength for analyzing TDF with suitable sensitivity.

 

Fig.2: Absorption spectra of TDF-PDAC-H+ system

 

Fig.3: Absorption spectra of TDF-HNO2-NED system

 

Preparation of calibration graph:

Method A: Aliquots of standard drug solution in methanol (1.0ml-3.0 ml, 200µg/ml) were placed in a series of 10ml calibrated tubes and volume of each test tube adjusted to 3.0ml with methanol. To each of these test tubes 1.0 ml of PDAC(6.31x 10-3M) and 1.0 ml of concentrated sulphuric acid (14M) were added, while cooling under a tap with constant shaking and kept in water bath at 60şc for 10min. cooled and diluted to the mark with methanol. The absorbance was measured at 502nm against the reagent blank within 10 minutes. The amount of drug in a sample was computed from Beer’s law plot (Fig.4).

 

Fig.4: Beer’s law plot of method A

 

Fig.5: Beer’s law plot of method B

 

Method B:

To aliquots of neutral solution of drug (1.0-3.0ml, 200µg/ml), 1.0ml of 2N HCl and 1.0ml of NaNO2 solutions were added successively into 25 ml calibrated tubes and kept aside for 15 min. after that 1.0ml of 2.5% aqueous solution of ammonium sulfa mate, 2.0ml 3M aqueous solution of sodium acetate and 1.0ml of 0.1% aqueous NED solutions were added successively. Purple colored species was formed and stable for 30 minutes. The absorbance was measured at 500nm against the similar reagent blank. The amount of drug in a sample was computed from its calibration graph (Fig. 5).  

 

RESULTS AND DISCUSSIONS:

In developing these methods, systematic studies of the effects of various parameters were undertaken by varying one parameter at a time and controlling all others fixed. The effect of various parameters such as time, temperature, nature and concentration of oxidant, volume and strength of reagents, order of addition of reagents on color development and solvent for final dilution on the intensity and stability of the colored species were studied and the optimum conditions were established.   The optical characteristics such as Beer’s  law limits, Sandell‘s sensitivity, molar extinction coefficient, percent relative standard deviation (calculated from the six measurements containing 3/4th of the amount of the upper Beer’s law limits)were calculated for both the methods and the results are summarized in table-1.Regression characteristics like standard deviation of slope (Sb), standard deviation of intercept (Sa), standard error of estimation (Se),% range of error (0.05 and 0.01 confidence limits) were calculated for both the methods and are shown in Table-1.

 

Commercial formulations containing TDF were successfully analyzed by the proposed methods. The values obtained by the proposed and reference methods for formulations were compared statistically by the t-and F-test and found not to differ significantly. As an additional demonstration of accuracy, recovery experiments were performed by adding a fixed amount of the drug to the pre analyzed formulations at three different concentration levels (80%, 100% and 120%). These results are summarized in Table-2.The ingredients usually present in formulations of TDF did not interfere with the proposed analytical methods.  Among the four aromatic aldehydes (vanillin, PDAC, PDAB and anisaldehydes) tried, all of them responded. But, PDAC was preferred as it was found to be better sensitivity in the assay of TDF for method A. Among three coupling reagents (phenol, α-Naphthol and NED) tried, all of them responded. But NED was preferred as it was found to be better sensitivity in method B.

 

Table  1: Optical characteristics, precision and accuracy of the proposed methods

Parameters

Method A

Method B

λ max(nm)

502

500

Beer’s law limit (µg/ml)

20-60  

8- 24

Sandell’s sensitivity (µg/cm2/0.001 abs. unit)

0.014814815

0.001823362

Molar absorptivity (Litre/mole/cm)

42897.6

348543

Regression equation (Y) *= a +b x

 

 

Intercept (a)

-0.099

-0.111

Slope(b)

0.009

0.029

%RSD

2.07

1.79

% Range of errors

(95% Confidence  limits)

0.05 significance level

0.01 significance level

 

 

2.17

 

 

1.89

3.41

2.96

*Y = a+ b x, where Y is the absorbance and x is the concentration of TDF in µg/ml

 

Chemistry of colored species:

In proposing method A, the nature of colored species formation with PDAC to form schiff base as it possess heterocyclic amino moiety. Bandelin and kemp 35 determined primary aromatic amines on the basis of diazotization and coupling with NED to yield a colored azodye and the same was adapted by Siggia 36 in quantitative organic analysis via functional groups. The presence of amino group in drug enables the diazotization with HNO2 and coupling with NED in acid medium to form colored species for method B and may be represented in schemes (Fig.6). 

 

 

Table 2: Analysis of TDF in pharmaceutical formulations

Method

*Formulations

Labeled Amount (mg)

Found by Proposed Methods

Found by Reference Method  ± SD

#% Recovery by Proposed Method ± SD

**Amount found ± SD

t

F

A

Batch-1

300

297.12±0.75

1.0

3.9

297.49 ±0.38

99.04±0.25

Batch-2

300

297.34±0.91

1.96

3.81

296.85 ± 1.78

99.11±0.30

B

Batch-1

300

296.29±0.84

1.55

4.86

297.49 ±0.38

98.76 ± 0.28

 

Batch-2

300

296.62  ±0.98

1.7

3.3

296.85 ± 1.78

98.87 ± 0.33

** Batch 1 and Batch 2 are tablets of different pharmaceutical companies of Tenof (Hetro) and Tavin (Emcure). 

 **Average ± Standard deviation of sis determinations, the t- and f-values refer to comparison of the proposed method with reference method. (UV). Theoretical values at 95% confidence limits t =2.57 and f = 5.05.

# Recovery of 10mg added to the pre analyzed sample (average of three determinations).

Reference method (reported UV method) using distilled water after dissolving in 1ml methanol

(גּ max=260nm).

 

 

Fig.6: Probable Schemes of method A and method B

 

 

CONCLUSION: 

The reagents utilized in the proposed methods are normal cost, readily available and the procedures do not involve any critical reaction conditions or tedious sample preparation. The proposed visible spectrophotometric methods are validated as per ICH guide lines and  possess reasonable precision, accuracy, simple, sensitive and can be used as alternative methods to the reported ones for the routine determination of TDF depending on the need and situation. 

 

ACKNOWLEDGEMENTS:

Author is grateful to University Grants Commission, New Delhi, for providing financial assistance under the Minor research project (Ref.no.F.MRP-3981/11) and   also very

much thankful to the m/s Tychy industries for providing gift sample of the drug.

 

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Received on 06.05.2013

Modified on 10.06.2013

Accepted on 22.06.2013     

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Research Journal of Pharmaceutical Dosage Forms and Technology. 5(4): July-August, 2013, 221-226