ISSN 0974-3618
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0974-360X (Online)
RESEARCH ARTICLE
A Simple
Validated UV Spectrophotometric Method for the Estimation of Tenofovir
disoproxil fumarate in bulk and Pharmaceutical dosage form
Mohammad Yunoos*, T. Durga Praveen,
P.S.N.C. Manikanta, Arjun Kumar Sai, V. Mounica, D. Siva
Department of Pharmaceutical Analysis, Bapatla College of
Pharmacy, Bapatla- 522101, Andhra Pradesh, India.
*Corresponding Author E-mail: yunoosvja@gmail.com
A
simple, accurate, rapid and sensitive UV spectrophotometric method has been
developed for the estimation of Tenofovir disoproxil fumarate (TDF) in bulk as
well as in Pharmaceutical dosage form. TDF is used as anti- retro-viral agent.
The present research work involves the estimation of TDF using methanol as
solvent. An absorption maximum (λ max) was found to be 259.5 nm. It obeys
the beer’s law in the concentration range of 5-50 µg/ml with a correlation
coefficient value of 0.9998. Validation of the method has been carried out
statistically as per ICH guidelines and the results were found to be
satisfactory. The result of mean % recovery was found to be 99.42 + 0.36 % for
TDF which shows that the method was not affected by the presence of common
excipients in tablet dosage form. The % RSD for both inter-day and intra-day
precision was found to be less than 2.0%. The limit of detection and limit of
quantification were found to be 0.47 µg/ml and 1.63 µg/ml respectively. The
method was validated by determining its sensitivity, linearity, accuracy and
precision which proves suitability of the developed method for the routine
quality control analysis for the estimation of Tenofovir disoproxil fumarate
(TDF) in bulk and tablet dosage form.
KEYWORDS: UV Spectrophotometry, Tenofovir disoproxil fumarate, Beer’s law,
validation, tablet dosage form.
INTRODUCTION:
Tenofovir
Disoproxil Fumerate, chemically, it is 9-[(R)-2
[[bis [[(isopropoxycarbonyl) oxy] methoxy] phosphinyl] methoxy] propyl] adenine
fumarate (1:1). Tenofovir
Disoproxil Fumerate (TDF) is achiral and has the empirical formula of C19H30N5O10P.C4H4O4
representing a molecular weight of 635.52gms/mol. It is a potent anti-retro
viral and belongs to a class of drugs known as
nucleotide reverse transcriptase inhibitors (NRTIs). Tenofovir is also used to treat a certain type of
liver infection called chronic hepatitis B infection. It helps to decrease the
amount of hepatitis B virus in your body by interfering with virus growth.
Before
phosphorylation, Tenofovir disoproxil fumarate is converted to tenofovir in the
intestinal lumen and plasma by diester hydrolysis. Tenofovir is
then internalized into cells, possibly by endocytosis, and subsequently
phosphorylated in sequential steps to tenofovir monophosphate and to the active
metabolite, tenofovir diphosphate. In a mechanism similar to that of NRTIs,
tenofovir diphosphate competes with its natural nucleotide counterpart,
deoxyadenosine 5´-triphosphate, for incorporation into newly forming HIV DNA1.
The review of literature revealed
that few analytical methods involving LC-MS/MS 2-3, LC-MS4 ,
HPLC5, and spectrophotometric methods6-8 in biological
fluids and in pharmaceutical dosage forms have been reported but no simple UV
Spectrophotometric method has been reported in pharmaceutical dosage forms so
far. The objective of this work is to develop a novel, simple, precise and rapid UV Spectrophotometric method for the
estimation of Tenofovir Disoproxil Fumerate in pharmaceutical dosage forms and
the developed method was validated in accordance with ICH guidelines and
which can successfully used for the assay of TDF in the Pharmaceutical dosage
form.
Figure: 1 Structure of Tenofovir
Disoproxil Fumerate (TDF)
MATERIALS
AND METHODS:
Instrumentation
An
ELICO UV-VIS double beam Spectrophotometer (SL 210 ) having Spectra Treats
(3.11.1) Software with a pair of 1cm matched quartz cells was employed to
measure absorbance of all solutions. Digital balance (Essae) of 1 mg
sensitivity was used for weighing.
Chemicals and Reagents
Tenofovir
Disoproxil Fumerate was received as a gift sample from Aurobindo Pharma Ltd.,
Hyderabad. The commercial tablet dosage form Tenvir manufactured by Cipla Ltd.,
Mumbai was procured from local pharmacy.
All reagents used were of analytical grade. Double distilled water (in house laboratory) was used in the following
experimental work.
Method Development
Preparation of standard and
linearity Solutions
Accurately
weighed 10 mg of reference standard of TDF was transferred into a 10 ml
volumetric flask and dissolved in methanol and then volume was adjusted up to
the mark with methanol to obtain a concentration of 1mg/ml. Further 1.0 ml of
the above stock solution was diluted to 10 ml in a 10 ml volumetric flask with
double distilled water to obtain working standard solution of concentration of
100 μg/ml. Aliquots was further diluted in 10 ml volumetric flasks to
obtain linearity solutions of concentration ranging from 5-50 µg/ml.
Preparation of sample solution
from tablet dosage form
20
tablets were accurately weighed and crushed to a fine powder. A quantity of the
tablet powder equivalent to 10 mg of TDF was accurately weighed and transferred
into a 10 ml volumetric flask and dissolved in methanol and then volume was
adjusted up to the mark with methanol. The resulting solution was filtered through
Whatmann filter paper no. 41. Further dilutions were carried out with double
distilled water to obtain a final concentration of 10µg/ml respectively.
Estimation of absorption maxima
of TDF
The absorption maxima of
Tenofovir Disoproxil Fumerate (TDF) was measured by scanning standard solution
of concentration 10µg/ml in the wave length range from 200-400nm. The λmax of TDF was found to
be 259.5nm as shown in figure 2. The absorbance of linearity solutions (5-50
µg/ml) were recorded at the selected wavelength (259.5nm) to obtain overlay
spectra as shown in figure 3.
RESULTS:
Method Validation
The developed method has been
validated as per ICH Q2 (R1) guidelines by means of the following parameters:
Linearity
The linearity was evaluated by
analyzing different concentrations of TDF linearity solutions. Appropriate
aliquots (0.1-1.0 ml) of TDF working standard solution (100µg/ml) were
transferred into the series of 10 ml volumetric flasks and the volume was made
up to the mark with double distilled water. The absorbance of the above
linearity solutions were measured at 259.5nm against double distilled water as
reagent blank. A calibration curve was constructed by plotting absorbance
versus concentration. The graph was found to be linear as shown in figure 4 and
regression equation was calculated. The results were shown in table 1.
Figure:
3 Calibration curve of TDF at 259.5nm
Table 1: Linearity Studies of TDF
|
S.No.
|
Parameters
|
TDF at 259.5nm
|
|
1
|
Beer’s law
limit (µg/ml)
|
5-50µg/ml
|
|
2
|
Regression
equation
|
Y=
0.0265x+0.0042
|
|
3
|
Correlation
coefficient (r)
|
0.99984
|
|
|
Intercept (a)
|
0.004218
|
|
|
Slope (b)
|
0.026531
|
Precision:
The repeatability of the proposed
method was ascertained by performing six independent assays of test sample. The
intra-day study was carried out on the same day at an interval of two hours
whereas inter-day study was carried out on three different days. The results of
statistical analysis were given in table 2.
Table 2: Precision Studies of TDF
|
Drug
|
Repeatability
(n=6)
% RSD
|
Inter-day Precision
(n=6) % RSD
|
Intra-day
Precision
(n=6) % RSD
|
|
TDF
|
0.62
|
0.57
|
0.76
|
Accuracy:
The accuracy of the proposed
method was determined by recovery studies using standard addition method. The %
recovery studies of TDF were carried out in three different concentration
levels (50%, 100% and 150%) by spiking previously analyzed samples of the
tablet with standard drug solutions. The results of accuracy studies and assay
were shown in table 3 and table 4.
Table 3: Accuracy Studies of TDF
|
Drug
|
%Spike level
|
% Recovery ± SD*
|
% RSD*
|
|
TDF
|
50
|
99.92.±0.02
|
0.36
|
|
100
|
98.84±0.076
|
0.48
|
|
150
|
99.49±0.069
|
0.23
|
*Mean
of three determinations
Table
4: Assay of Tenofovir Disoproxil Fumerate (TDF) in Formulation
|
Drug
|
Labeled Amount
(mg)
|
Amount found
(mg)
|
% Assay
|
|
TDF
|
300
|
298.9
|
99.63
|
The optical characteristics
such as Beer’s law limit, molar absorptivity, Sandell’s sensitivity,
Correlation coefficient, slope and intercept, % Relative Standard Deviation
(Precision), Limit of detection and limit of quantification were calculated and
are summarized in table 5.
Table 5: Optical characteristics and Precision of proposed UV
Spectrophotometric method for TDF
|
Parameter
|
Results
|
|
λ max (nm)
|
259.5
|
|
Beer’s law
limit (mg/mL)
|
5-50
|
|
Molar
absorptivity (L mole-1 cm-1)
(L mole-1
cm-1)
|
1.462x104
|
|
Correlation coefficient (r)
|
0.99984
|
|
Slope (b)
|
0.026531
|
|
Intercept (a)
|
0.004218
|
|
Regression
equation (Y = a + bC) **
|
0.0265x+0.0042
|
|
Repeat (%RSD)*
Relative
standard
deviation
deviation (%)*
|
0.62
|
|
Inter-day
precision (%RSD)*
|
0.57
|
|
Intra-day
precision (%RSD)*
|
0.76
|
|
Sandell’s
sensitivity
(mg cm-2
/ 0.001 Abs unit)
|
0.0042
|
|
Limit of
Detection (LOD)
|
0.47µg/ml
|
|
Limit of
Quantitation (LOQ)
|
1.63µg/ml
|
*Mean of six determinations
DISCUSSIONS:
In this proposed method the
absorbance of all solutions were measured at 259.5nm against double distilled
water as reagent blank. TDF obeyed beer’s law in the concentration range of
5-50 µg/ml with correlation coefficient (r) of 0.99984. %RSD for inter-day and
intra-day precision were found to be 0.57 and 0.76, respectively. %RSD for
repeatability was found to be 0.62 for TDF. As the %RSD values were below 2%,
the method was found to be highly precise. %Recovery values of TDF were found
to be 98.84-99.92 % respectively which indicates that the proposed method is
accurate. Tablet dosage form was analyzed and the result of assay of the drug
was in good agreement with the label claim of formulation as indicated by %
assay with 99.63% for TDF. All the results were found to be within the limits
and therefore the proposed method was found to be free from interferences from
excipients from the tablet dosage form.
CONCLUSION:
A simple, sensitive, rapid and
economic UV spectrophotometric method has been developed and validated for the
assay Tenofovir Disoproxil Fumerate (TDF) in tablet formulation. This method
yielded high recoveries with good linearity and precision. It can be concluded
that the proposed method is a good approach for obtaining reliable results and
found to be suitable for the routine analysis of TDF in tablet formulation.
ACKNOWLEDGEMENT:
The author is thankful to
Bapatla College of Pharmacy for providing facilities required for the research.
The author is also thankful to Aurobindo Pharma Ltd., Hyderabad for providing the gift sample of Tenofovir
Disoproxil Fumerate.
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